EP2030021B1

EP2030021B1 - Method for diagnosing mitochondrial dysfunction

Info

Publication number: EP2030021B1
Application number: EP07725374A
Authority: EP
Inventors: Andreas Reichert; Stéphane DUVEZIN-CAUBET; Johannes Wagener; Sabine Hofmann-Thiel; Walter Neupert
Original assignee: Individual
Current assignee: Reichert Andreas
Priority date: 2006-05-18
Filing date: 2007-05-18
Publication date: 2012-11-21
Anticipated expiration: 2027-05-18
Also published as: WO2007134818A2; EP2030021A2; WO2007134818A3

Description

The present invention relates to means and methods for the diagnosis of mitochondrial disorders/dysfunctions, in particular to a method for determining the susceptibility for, predisposition for or the presence of a disorder correlated with mitochondrial dysfunction or mitochondrial disease, wherein said method comprises the in vitro determination of the ratio of large and/or small isoforms of the optic atrophy 1 protein (OPA1). Furthermore, uses and kits for carrying out these methods are disclosed.
Mitochondria are essential organelles in eukaryotic cells. They perform a plethora of functions and interact with the rest of the cell in a multitude of pathways. Growth, differentiation and maintenance of cells as well as development of tissues and organs is dependent on an adaptive development of mitochondria. This is also apparent from the occurrence of a vast number of human disorders in which mitochondrial function is compromised due to genetic alterations, toxic effects or stress.
The most conspicuous function of mitochondria is in the energy metabolism of the cell. Oxidative phosphorylation is by far the most important pathway of synthesizing ATP and thereby maintaining life. Control of energy metabolism, tissue specific regulation of components of the respiratory chain and ATP synthase are central to tissue performance and therefore an issue in numerous diseases.
Mitochondria have the cellular monopole in housing a considerable number of anabolic pathways leading to the formation of essential components for the cell. Examples are the biosynthesis of haem, of amino acids and of iron-sulfur clusters. In addition, mitochondria harbour essential catabolic pathways such as degradation of fatty acids.
Mitochondria are prominent producers of reactive oxygen species whose, mainly damaging, roles are at the base of numerous processes leading to disease. One of these processes is mutation of mitochondrial DNA promoting defects of oxidative phosphorylation and tissue damage. Damage to mitochondrial DNA has been shown to be causative to premature ageing in mice. Therefore, it is of utmost importance, e.g. for the development of stem cells and generations of engineered tissues, to minimize mitochondrial damage, e.g. due to reactive oxygen species.
Mitochondria play a central role in apoptosis. Apoptosis is a useful and necessary process when it comes to development and destruction of cancer tissue. However, apoptosis can also be responsible for undesired breakdown of cells and tissues.
Mitochondria are highly motile organelles. This property is required for positioning mitochondria in polarized cells, most conspicuously in nerve cells. Mitochondria are also dynamic in terms of continuously occurring and rapid events of fusion and fission. This process is determining mitochondrial structure. Mitochondria form a highly interconnected network in the cell. The integrity of the network is required for mitochondrial competence and for prevention of apoptosis.
Many human diseases are caused or accompanied by mitochondrial dysfunction arising from mutations in mitochondrial or nuclear DNA. Mutations in mitochondrial DNA (mtDNA) affecting essential subunits of the respiratory chain or mitochondrial tRNA genes are associated with common mitochondrial diseases such as MELAS (Myopathy encephalopathy lactic acidosis and stroke-like episodes), myoclonic epilepsy with ragged-red fiber syndrome (MERRF syndrome), cardiomyopathy, Leber's hereditary opticus neuropathy (LHON), Leigh's syndrome, etc., to mention just a few of them (reviewed in Wallace, 2005, Annu Rev Genet, 39, 359-407). Mutations, deletions and insertions in mtDNA have been detected in numerous types of cancer such as renal cell and colorectal carcinoma, early liver, protasta, breast, bladder, primary lung, head and neck tumours, astrocytomas, adenocarcinomas in Barrett's esophagus (Alonso, 1997, Electrophoresis, 18, 682-685; Fliss, 2000, Science, 287, 2017-2019; Habano, 1999, Int J Cancer, 83, 625-629; Horton, 1996, Genes Chromosomes Cancer, 15, 95-101; Jeronimo, 2001, Oncogene, 20, 5195-5198; Kirches, 1999, Genes Chromosomes Cancer, 26, 80-83; Miyazono, 2002, Oncogene, 21, 3780-3783; Modica-Napolitano, 2004, Mitochondrion, 4, 755-762; Parrella, 2001, Cancer Res, 61, 7623-7626; Polyak, 1998, Nat Genet, 20, 291-293; Tan, 2002, Cancer Res, 62, 972-976). Moreover, the occurrence of these mutations appear to be several-fold increased in tumour versus control tissues (Fliss, 2000, Science, 287, 2017-2019; Horton, 1996, Genes Chromosomes Cancer, 15, 95-101). Moreover, a major contributing factor to Parkinson's as well as Alzheimer's disease is the reduced cellular ATP level caused by mitochondrial dysfunction. (Hashimoto, 2003, Curr Biol, 14, 340-345; Orth, 2002, Neurochem Int, 40, 533-541). Impairment of glucose and fatty acid metabolism due to mitochondrial dysfunction are important factors in diabetes and obesity, respectively (Ballinger, 1994, Nat Genet, 7, 458-459; Ballinger, 1992, Nat Genet, 1, 11-15; Gerbitz, 1996, Diabetes, 45, 113-126). Interestingly, the level of mutated mtDNA is also increasing with age - also for non-symptomatic humans. In a transgenic mouse, expression of an error-prone gamma mtDNA polymerase led to mtDNA mutations during mtDNA replication and those mice showed a clear phenotype of premature ageing (Trifunovic, 2004, Nature, 429, 417-423). This study showed that accumulation of mtDNA mutations is causative to ageing.
Mitochondrial morphology appears to be of utmost importance for the maintenance of mitochondrial function. In many cell types, mitochondria form a large network of interconnected tubules that is maintained by a balance of fission and fusion events (Nunnari, 1997, Mol Biol Cell, 8, 1233-1242). The physiological role of this dynamic and energy-consuming process is unclear. Apoptosis requires DRP1-dependent mitochondrial fission (Breckenridge, 2003, J Cell Biol, 160, 1115-1127; Frank, 2001, Dev Cell, 1, 515-525; Jagasia, 2005, Nature, 433, 754-760; Lee, 2004, Mol Biol Cell, 15, 5001-5011). The number and plasticity of dendritic spines and synapses and the mobilization of reserve pool vesicles in synapses depend on the balance between fusion and fission of mitochondria (Li, 2004, Cell, 119, 873-887; Verstreken, 2005, Neuron, 47, 365-378). Mitochondrial fusion was proposed to counteract the effects of mutations in mtDNA by trans-complementation, and therefore to impede the development of mitochondrial diseases (Nakada, 2001, Nat Med, 7, 934-940; Ono, 2001, Nat Genet, 28, 272-275). LETM1 is deleted in the Wolf-Hirschhorn syndrome (Nowikovsky, 2004, J Biol Chem, 279, 30307-30315; Schlickum, 2004, Genomics, 83, 254-261). The yeast orthologue Mdm38 was shown to be essential for wildtype mitochondrial morphology (Dimmer, 2002, Mol Biol Cell, 13, 847-853). Recently, mutations in the ganglioside induced protein 1 that promotes formation of elongated tubular mitochondria were associated with Charcot-Marie-Tooth disease type 4a (Niemann, 2005, J Cell Biol, 170, 1067-78).
Two proteins involved infusion of mitochondria, OPA1 (optic atrophy 1 protein) and Mitofusin2, are known to be affected in optic atrophy type 1 and Charcot-Marie-Tooth disease type 2a, respectively (Alexander, 2000, Nat Genet, 26, 211-215; Delettre, 2000, Nat Genet, 26, 207-210; Zuchner, 2004, Nat Genet, 36, 449-451). Overexpression of OPA1 in mouse embryo fibroblasts promotes tubulation of mitochondria (Cipolat, 2004, Proc Natl Acad Sci USA). Yet, downregulation of OPA1 leads to fragmentation of mitochondria, mitochondrial dysfunction, altered mitochondrial inner membrane morphology and increased propensity to apoptosis (Chen, 2005, J Biol Chem, 280, 26185-26192; Griparic, 2004, J Biol Chem, 279, 18792-18798; Lee, 2004, Mol Biol Cell, 15, 5001-5011; Olichon, 2003, J. Biol. Chem., 278, 7743-7746). The prior art observed fragmentation of mitochondria in over- as well as down-regulation experiments of OPA1 and the corresponding function of OPA1 remains obscure.
Eight alternatively spliced mRNAs transcribed from the OPA1 gene (OPA1 spliceforms 1 to 8) and tissue- and cell line-specific protein isoforms were described ( WO 02/00878 ; Delettre, 2001, Hum Genet, 109,584-591; Olichon, 2002, FEBS Lett, 523,171-176; Satoh, 2003, Biochem Biophys Res Commun, 300, 482-493). The ortholog of OPA1 in baker's yeast, Mgm1, was shown to be essential for mitochondrial fusion (Sesaki, 2003, Mol Biol Cell, 14, 2342-2356; Wong, 2003, J Cell Biol, 160, 303-311). It was demonstrated that Mgm1 is present in two isoforms in the intermembrane space of mitochondria (Herlan, 2003, J Biol Chem, 278, 27781-27788; Wong, 2000, J Cell Biol, 151, 341-352). The exact N-termini of both isoforms were determined with biochemical means and it was found that Mgm1 biogenesis depends on the novel mitochondrial rhomboid protease Pcp1 (Herlan, 2003, J Biol Chem, 278, 27781-27788). Both Mgm1 isoforms appear to be required in roughly equal amounts in order to maintain mitochondrial morphology and mtDNA. Mgm1 is partially cleaved by Pcp1 within a second, so far unrecognized conserved, hydrophobic segment of Mgm1 (Herlan, 2004, J Cell Biol, 165, 167-173). Mutations within the first hydrophobic segment of Mgm1 led to alterations of the ratio of the two isoforms and to fragmentation of mitochondria. It was further shown that formation of the short isoform (s-Mgm1) and mitochondrial morphology depend on a functional import motor and on the ATP level in the matrix. In an atp6 mutant, impaired in mitochondrial ATP synthesis, the formation of s-Mgm1 is reduced. Therefore, mitochondrial morphology depends on an ATP-dependent mechanism which was termed "alternative topogenesis" (Henan, 2004, J Cell Biol, 165, 167-173).
In yeast, any mitochondrial dysfunction that leads to reduced ATP levels in the matrix, would in tum lead to a reduced formation of s-Mgm1, while I-Mgm1 is not affected. The consequence is that such mitochondria cannot fuse anymore with the intact mitochondrial network and are therefore excluded from inheritance to daughter cells.
A Drosophila melanogaster homolog of OPA1 (CD8479) is known to enhance the activity of the Drosophila melanogaster Uncoupling Protein (dUCPy), the overexpression of which is assumed to be lethal for the corresponding transgenic flies due to a collaps of the cellular energy production.- WO 03/061681
In humans, many muscular and neurological disorders, various forms of cancer, diabetes, obesity, other disorders and ageing are associated with mitochondrial dysfunction as discussed above and in Wallace, 2005, Annu Rev Genet 39, 359-407, Modica-Napolitano, 2004, Mitochondrion 4, 755-62 or Orth, 2001, Am J Med Genet 106, 27-36). Deficiencies in mitochondrial fusion and fission, two opposing processes that determine mitochondrial morphology (Nunnari, 1997, Mol Biol Cell 8, 1233-42; Okamoto, 2005, Annu Rev Genet 39, 503-36), play an important role in such disorders, in particular in various neurodegenerative disorders (Alexander, 2000, Nat Genet 26, 211-5; Delettre, 2000, Nat Genet 26, 207-10; Zuchner, 2004, Nat Genet 36, 449-51; Niemann, 2005, J Cell Biol 170, 1067-78). Mutations in the OPA1 gene, the human ortholog of Mgm1, are known to cause hereditary autosomal dominant optic atrophy type I, the most prevalent neuropathy of the optic nerve occurring with a prevalence of 1:12000 to 1:50000 dependent on the population (Alexander, 2000, Nat Genet 26, 211; Delettre, 2000, Nat Genet 26, 207). Seven particular mutations of the OPA1 gene were identified in patients suffering from autosomal dominant optic atrophy ( WO 02/27022 ). In addition, 14 mutations which affect the activity of the OPA1 isoforms were identified in humans suffering from optic atrophy and a screening method was developed, wherein the OPA1 isoforms are applied to find modulators of the activity of the OPA1 isoforms to treat diseases that are associated with mitochondrial dysfunction ( WO 02/00878 ). OPA1 appears to be required for mitochondrial fusion (Olichon, 2003, J. Biol. Chem. 278; Cipolat, 2004, Proc Natl Acad Sci USA; Chen, 2005, J Biol Chem 280, 26185). Overexpression of OPA1 in mouse embryo fibroblasts promotes tubulation of mitochondria (Cipolat, 2004, Proc Natl Acad Sci USA, 101, 15927-32). Downregulation of OPA1 leads to fragmentation of mitochondria, mitochondrial dysfunction, altered mitochondrial inner membrane morphology and increased propensity for apoptosis (Lee, 2004, ; Olichon, 2003, J. Biol. Chem. 278, 7743; Chen, 2005, J Biol Chem 280, 26185; Griparic, 2004, J Biol Chem 279, 18792).
OPA1, is known to be transcribed into eight alternative splice variants which vary in the presence or absence of exon 4, 4b, and/or 5b (see protein sequence alignment in Fig.1). The expression of these splice variants is reported to vary among different tissues. In addition, these splice variants give rise to several protein isoforms (Alexander, 2000, Nat Genet, 26, 211-215; Delettre, 2001, Hum Genet, 109, 584-591; Delettre, 2000, Nat Genet, 26, 207-210; Delettre, 2002, Mol Genet Metab, 75, 97-107; Griparic, 2004, J Biol Chem, 279, 18792-18798; Olichon, 2003, J. Biol. Chem., 278, 7743-7746; Olichon, 2002, FEBS. Lett, 523, 171-176; Pesch, 2004, Invest Ophthalmol Vis Sci, 45, 4217-4225; Pesch, 2001, Hum Mol Genet, 10, 1359-1368; Satoh. 2003, Biochem Biophys Res Commun, 300, 482-493; Thiselton, 2002, Invest Ophthalmol Vis Sci, 43, 1715-1724). The identity and the biogenesis of the different protein isoforms of OPA1 remain elusive in the prior art and are not known.
Diagnosis of mitochondrial dysfunction has so far been based on the following detection systems. Various DNA-based detection/screening methods for nuclear or mitochondrial DNA mutations are commonly used. For example, there are methods to determine enzymatic activities of mitochondria, such as enzymatic activities of the respiratory chain or F₁F_O-ATP synthase and various histological methods allowing staining of enzymes of the respiratory chain complexes. Evaluation of the ultrastructure of mitochondria is done by electron microscopy. These methods are very laborious, difficult to automate, too specialized (for only single defects), not robust, not easy to perform, or require large amounts and difficult to get tissue samples. Methods employed in the art mostly determine isolated mitochondrial activities and do not allow a simple and general evaluation of the functionality of mitochondria. Mostly a variety of methods have to be performed and evaluated in a combined fashion to give a general diagnostic overview of mitochondrial function.
The technical problem underlying the present invention is the provision of means and methods for diagnosing mitochondrial dysfunction and diseases related thereto, respectively. In particular, means and methods for the early detection of mitochondrial dysfunction and diseases/disorders related thereto are of need.
The solution to the above technical problem is achieved by providing the embodiments characterized in the claims.
The present invention relates to a method for determining the susceptibility for, predisposition for or the presence of a disorder correlated with mitochondrial dysfunction or mitochondrial disease, comprising the in vitro determination selected from the group consisting of

(a) the in vitro determination of the ratio of at least one large and at least one small isoform of the optic atrophy 1 protein (OPA1), whereby said large isoform has an apparent molecular weight of more than 91 kD and whereby said small isoform has an apparent molecular weight of less than 91 kD, said molecular weights being determined by SDS-PAGE analysis; and/or
whereby said large isoform has an apparent molecular weight of more than 95 kD and whereby said small isoform has an apparent molecular weight of less than 95 kD, said molecular weights being determined by mass spectrometry;
(b) the in vitro determination of (a) wherein the ratio of at least two OPA1 isoforms is determined; and
(c) the in vitro determination of (a) comprising the step of determining the ratio between at least one large OPA1 isoform and an internal standard and between at least one small OPA1 isoform and an internal standard,

The present invention solves the above identified technical problem since, as documented herein below and in the appended examples, it was surprisingly found that OPA1 plays a certain role in a pathway selecting against dysfunctional mitochondria. As documented in the appended examples, a (proteolytically) processing of large OPA1 isoforms into small OPA1 isoforms indicates a predisposition for, a susceptibility for or the presence of a disorder correlated with mitochondrial dysfunction. In particular, in context of the present invention, it was found that mitochondrial fragmentation occurs concomitant with processing of OPA1. This happens under various conditions of mitochondrial dysfunction and, e.g., during apoptosis. The proteolytic cleavage or convertion of large OPA1 isoforms into smaller isoforms, in particular (but not limiting) into a small OPA1 isoform denoted as "OPA1-S5", -S5-OPA1-, "OPA1#5" or "s-OPA1#5" herein, is a diagnostic tool for the determination of a mitochondrial disorder/disease which can lead to a manifestation of a corresponding clinical and/or pathological situation, like (but not limiting) neurological disorders (e. g. Alzheimer's disease, bipolar disorders, stroke, Charcot-Marie-Tooth disease, ALS or Parkinson's disease), myopathies (e. g. general myopathy, Ataxia, infantile myopathy, atrophies, ocular myopathy, motor neuron disorders, general encephalomyopathy, Leigh-Syndrom, MELAS (myopathy encephalopathy lactic acidosis and stroke-like episodes), MERRF (myoclonic epilepsy with ragged-red fiber disease) or optic atrophy type 1), metabolic disorders (e. g. diabetes or obesity), infection disorders (e. g. bacterial, fungal or viral infections), neoplastic disorders or cancers, ischemias, oxidative damages and the like.
Further disorders associated with a mitochiondrial dysfunction and to be detected (for example in vitro) by the means and methods provided herein are given herein below.
As mentioned above, the present invention relates to a method for determining the susceptibility for, predisposition for or the presence of a disorder correlated with mitochondrial dysfunction or mitochondrial disease, comprising the in vitro determination selected from the group consisting of

In particular, said large isoform(s) has (have) an apparent molecular weight of more than 91 kD and said small isoform has an apparent molecular weight of less than 91 kD, when said molecular weights being determined by SDS-PAGE analysis, in particular an 10% gel as disclosed herein and described in the appended examples. It is evident for the person skilled in the art that also other SDS gels and means (in particular Western-Blot analysis and the like) are useful and envisaged in context of the present invention. It is of note that the herein given value of 91 kD is, accordingly, an illustrative example and the person skilled in the art can also use other means to deduce the ratio of the herein described OPA isoforms
in a given sample to be analysed. For example, said large OPA1 isoform(s) has (have) an apparent molecular weight of more than 95 kD or, preferably, of more than 99 kD and the small OPA1 isoform(s) has (have) an apparent molecular weight of less than 95 kD or, preferably, of more than 99 kD, when said molecular weights being determined by peptide analysis, e.g. mass spectrometry.
As mentioned herein, in context of the present invention, "OPA" or "OPA1" means the optic atrophy 1 protein/gene, in particular OPA1 of human origin. Yet, in certain embodiments it is also envisaged that OPA1 of other organisms, e. g. of mouse, rat, pig, dog, bovine species or fruit fly, be assessed in context of this invention. The nucleotide and amino acid sequences of human OPA1, particularly of the eight spliceforms of OPA1, are given in the appended sequence listing and examples.
It is of note that the nucleotide and amino acid sequences of OPA1 given herein below are not limiting. Accordingly, the term "OPA" or "OPA1" also encompasses OPA1 proteins/genes having amino acid or nucleotide sequences being derivatives of those given sequences.
In terms of the present invention the term "derivatives" or "derivatives thereof" refers to amino acid or nucleotide sequences being homologous to the amino acid or nucleotide sequences shown herein, e. g. those of human OPA1, and/or amino acid or nucleotide sequences as shown herein, e. g. those of human OPA1, but having (a) particular conservative amino acid(s) exchanged. For instance, in context of the present invention, "homologous" means that amino acid or nucleotide sequences have identities of at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% to the sequences shown herein, e. g. those of human OPA1, wherein the higer identity values are preferred upon the lower ones.
As shown herein, upon drug-induced apoptosis, processing of OPA1 and mitochondrial fragmentation preceeded cytochrome c release. When the mitochondrial membrane potential was dissipated, processing of OPA1 and fragmentation of mitochondria, but not cytochrome c release was observed. The same phenomenon was observed in cybrid cells from a patient with MERRF syndrome and in mouse embryonic fibroblasts harbouring an error-prone mitochondrial DNA (mtDNA) polymerase gamma. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM -/- knockout mice suffering from mitochondrial cardiomyopathy and in skeletal muscle from patients with mitochondrial disorders such as MELAS. Processing of OPA1 was inhibited by addition of ortho-phenantroline and partially by addition of DCl (3,4-Dichloroisocoumarin) in vivo.
Recovery of mitochondrial fusion was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria could be prevented by overexpressing OPA1.
This demonstrates that various forms of mitochondrial dysfunction lead to proteolytic processing of OPA1 resulting in impaired mitochondrial fusion and points out to the existence of a pathway in which processing of OPA1 leads to fragmentation of mitochondria. Without being bound by theory, this fragmentation then serves as an early response in order to segregate dysfunctional mitochondria from the network of functional mitochondria.
These surprising findings demonstrate a proteolytic processing of larger OPA1 isoforms into smaller ones and a corresponding relationship to mitochondrial dysfunction. Accordingly, said processing occurs under various conditions of mitochondrial dysfunction, said dysfunction being linked to pathological status.
The processes of mitochondrial damage leading to dysfunction, breakdown of mitochondrial bioenergetic competence and mitochondrial fragmentation are linked through a cascade of reactions. In context of the present invention, a central molecular player, namely OPA1, that links changes in mitochondrial morphology with mitochondrial dysfunction was identified. This result is exemplary based on a wide variety of established model systems and patient material for, e. g., MERRF, MELAS, mtDNA depletion syndrome, dilated cardiomyopathy, diseases with respiratory deficiencies, e. g. diseases with respiratory deficiencies of unknown origin and aging. Without being bound by theory, it can be deduced how this cascade is organized: Mitochondrial dysfunction leads to impairment of bioenergetic competence of mitochondria. This results in reduced membrane potential and ATP production. In such compromised mitochondria a proteolytic processing of large to small isoforms of OPA1 is activated. As a consequence, fusion of mitochondria is blocked and dysfunctional mitochondria are segregated from the network of intact mitochondria. This in tum triggers further reactions such as removal and degradation of the dysfunctional fragments as reported in several systems (Priault, 2005, Cell Death Differ, online publication, 10 June 2005; Lyamzaev, 2004, ; Skulachev, 2004, Mol Cell Biochem 256-257, 341). A key element of this mechanism is the regulatory inactivation of fusion-promoting OPA1 by proteolytic cleavage.
It was found in the art that, in contrast to OPA1, in the yeast homolog of OPA1 (Mgm1), lack of either the large or the small isoforms leads to an impairment of mitochondrial fusion (Herlan, 2003, J Biol Chem 278, 27781; Herlan, 2004, J Cell Biol 165,167; Sesaki, 2003, Biochem ). Accordingly, it was speculated, that a proper balance of long and short isoforms of Mgm1 is critical for maintainance of tubular mitochondrial morphology and that, in analogy therto, there is also the possibility that an overexpression of OPA1 can lead to an imbalance in OPA1 isoforms (Chen, 2005, J Biol Chem 280, 26185)
Yet, based on the teaching provided in context of this invention, mitochondrial dysfunction (or a corresponding mitochondrial disease) is not merely correlated with decrease of any one of OPA1 isoforms, but with a decrease of particulary the large isoforms, e. g. OPA1#1 (as defined herein) and OPA1#2 (as defined herein), accompanied by an increase of the small isoforms, e. g. OPA1#3 (as defined herein) and OPA1#5 (as defined herein).
In context of the present invention it was found that the described large and small isoforms of yeast Mgm1 do not correlate to large and small isoforms of OPA1. Accordingly, the above mentioned finding that mitochondrial fragmentation in humans is correlated with a decrease of particularly large isoforms of OPA1 and an an increase of particulary small isoforms of OPA1 was unexpected.
In yeast, mitochondrial dysfunction causes a deficiency in the import of the Mgm1 precursor. Consequently, formation of the small isoform and mitochondrial fusion are impaired (Herlan, 2004, J Cell Biol 165, 167). In humans, mitochondrial dysfunction is sensed in a different fashion which is independent of protein synthesis and consequently protein import into mitochondria. Still, this leads to the specific, rapid, and intramitochondrial inactivation of the orthologous effector protein, OPA1. Taken together, mitochondrial dysfunction leads to a rapid conversion of the large isoforms into the small isoforms in humans whereas in yeast this process occurs in the opposite direction (increase of large isoform) and is slow since it requires protein turnover.
Satoh (2003, Biochem, Biophys Res Commun, 300, 482-493) identified 2 OPA1 isoforms in HeLa cells and speculates that their differential association with the inner and outer mitochondrial membrane suggests the different roles of each of these proteins for controlling the mitochondrial morphology. It is not evident from Satoh that a specific pattern of occurrence of different OPA1 isoforms, not to mention the herein described decrease of large accompanied with an increase of small isoforms, correlates with the fragmentation status of mitochondria.
As mentioned above, herein provided and demonstrated by the appended examples is a method for determining the susceptibility for, predisposition for or the precence of a disorder correlated with mitochondrial dysfunction or mitochondrial disorder/disease.
In context of the present invention, the term "correlated with" means that either a certain clinical and/or pathological situation causes mitochondrial dysfunction or that mitochondrial dysfunction leads to a certain clinical and/or pathological situation. It is of note that the present invention is particularly useful in the determination of a mitochondrial disease, dysfunction or disorder before any clinical and/or pathological symptoms are diagnosed or determined by the attending physician. Accordingly, the present invention is particularly usefull in early determination/diagnosis of a mitochondrial dysfunction and/or correlated disease.

In addition or in iteration to the above, clinical and/or pathological situations correlated with mitochondrial dysfunction or mitochondrial disorder/disease are given in the table below.

Ageing: in particular pathological and/or pre-mature aging

Alzheimer's disease

Amyotrophic lateral sclerosis (ALS)

Apoptosis

Ataxia

Autism

Barth syndrome, (familial)

Bipolar disorder

Cancer (e.g. renal cell and colorectal carcinoma, early liver, protasta, breast, bladder, primary lung, head and neck tumours, astrocytomas, adenocarcinomas in Barrett's esophagus)

Cardiomyopathy

Charcot-Marie-Tooth disease type 2a

Charcot-Marie-Tooth disease type 4a

Congenital lactic acidosis

Crohn disease

Deafness

Diabetes

Diabetic sensory neuropathy

Encephalomyopathy

Endotoxemia

External ophthalmoplegia (e.g. PEO)

Friedreich's ataxia

Hepatopathy (e.g. defects in SCO1)

Hepato-cerebral form of mtDNA depletion syndrome

Hereditary sensory neuropathy

Hereditary spastic paraplegia

Infantile encephalopathy

Infantile myopathy

Infectious diseases

Inflammatory diseases

Ischemia-reperfusion injury / Hypoxic damaqe / Oxidative damaqe

Kearns-Sayre syndrome

Lactic acidosis

Leber's hereditary opticus neuropathy (LHON)

Leigh's syndrome

Leukodystrophy

Mohr-Tranebjaerg-syndrome

Metabolic disorders (e.g. defective glucose and fatty acid metabolism)

Mitochondrial neurogastrointestinal-encephalomyopathy

Motor neuron disorders

mtDNA depletion syndrome

Myoclonus epilepsy and ragged-red fibers syndrome (MERRF)

Myopathy

Myopathy encephalopathy lactic acidosis and stroke-like episodes (MELAS)

Myositis

Neurodegenerative disorders (e.g. autosomal dominant optic atrophy, Charcot-Marie-tooth disease, Wolf-Hirschhom syndrome, ALS)

Non-alcoholic fatty liver disease

Obesity

Ocular myopathy

Optic atrophy type 1

Paraganqlioma (e.g. defects in complex II / SDH)

Parkinson's disease

Pearson's syndrome

respiratory chain disorder

Rhabdomyolysis

Schizophrenia

Sideroblastic anemia

Stroke

Tubulopathy (e.g. defects in BCS1L)

Viral and bacterial infections

Wolfram syndrome

The terms "disorder related to mitochondrial dysfunction", "mitochondrial disorder" or "mitochondrial disease" is not to be construed to the disorders provided above.
In context of the present invention, a certain given molecular weigth value , means +/- 3 kD, preferably +/- 2 kD, more preferably +/- 1 kD, more preferably +/- 0.5 kD and most preferably +/- 0.1 kD of said value. Moreover, in context of the present invention, it is envisaged that the term "less than xx kD", for example "less than 91 kD". "less than 95 kD" or "less than 99 kD", also comprises molecular weigth values being equal to xx kD, for example equal to 91, 95 kD or 99 kD.
It is evident for the person skilled in the art that certain given molecular weight values may vary, dependent on the preparational/experimental conditions employed, or, for example with respect to mass spectrometry, dependent on the information content resulting from the preparational/experimental method employed or dependent on an employed modification of the proteins/peptides to be analyzed due to a specific preparational/experimental procedure. It is, for example, known in the art that proteins/peptides to be analyzed via mass spectrometry can be modified, i. e. their theoretical molecular weight can be increased (e.g. by certain chemical modifications) or decreased (e.g. by using (a) certain protease(s)) by a certain value. It is therefore of note in context of the present invention that the molecular weigth values given for certain OPA1 isoforms can change, dependent on the particular preparational/experimental conditions employed during the corresponding mass spectrometry experiment (or other methods for determining molecular weights). The skilled person is readily in the position to deduce whether certain changes/differencies of given molecular weight values result from the particular preparational/experimental method employed or form a specific composition of the protein/peptide analysed.
In context of the present invention, the term "isoform" of OPA1 means a certain form of the OPA1 protein. Without bound by theory, an OPA1 isoform derives from (a protein encoded by) any one of spliceforms 1 to 8 of OPA1, e.g. by posttranslational processing (e. g. proteolytical processing). Without bound by theory, said posttranslational processing (e. g. proteolytical processing) leads to a shortened N-terminus of OPA1, particularly of spliceforms thereof, wherein the C-terminus remains complete. The "isoforms" of OPA1 to be scrutinized in context of the present invention are described herein in more detail. Accordingly, the term "corresponding" in context of OPA1 isoforms and OPA1 spliceforms, e.g. in the term "an OPA1 isoform having an apparent molecular weight calculated from amino acid sequences of the corresponding spliceform(s)", means that the respective OPA1 isoform can be related to or may be derived from said OPA1 spliceform(s). These spliceforms are also described herein below.
In context of the present invention, the term "spliceform" or "splice variant" of OPA1 means a form of OPA1 that emerges by alternative splicing of the primary transcript transcribed from the OPA1 gene. It is envisaged herein, that the term "spliceform" either refers to the mature transcript generated by alternative splicing, but also refers to the corresponding protein which has been translated from said mature transcript. Accordingly, the term "isoform being derived from (correponding) spliceform" means that an OPA1 isoform originates from a protein that has been translated from a mature (alternatively spliced) transcript of the OPA1 gene. Thereby, posttranslational processing (e. g. proteolytical processing) of said protein that has been translated from a mature (alternatively spliced) transcript of the OPA1 gene may occur. However, an OPA1 isoform may also directly originate from said protein, without further posttranslational processing. In such specific case, said protein then is said OPA1 isoform.
At present, 8 spliceforms of OPA1 are known in the art, which emerge by alternative splicing of exon 4, exon 4b and/or exon 5 (see Fig. 1). The corresponding amino acid sequences of these 8 spliceforms are given in SEQ ID No: 2, 4, 6, 8, 10, 12, 14 and 14 and are schown in Fig. 1. Their corresponding nucleotide acid sequences are given in SEQ ID No: 1, 3, 5, 7, 9, 11, 13 and 15.
Dependent whether exon 4, exon 4b and/or exon 5 is comprised, the OPA1 spliceforms can be defined by specific amino acid sequences, e. g. by one of the following amino acid sequences:

EYKWIVPDIVWEIDEYIDFGHKLVSEVIGASDLLLLL (SEQ ID NO: 31) corresponds to the amino acid sequences from exon 3 to exon 4b (lack of exon 4) and is comprised in spliceforms 3 and 6.
EYKWIVPDIVWEIDEYIDFGSPEETAFRATDRGSESDKHFRK (SEQ ID NO: 32) corresponds to the amino acid sequences from exon 3 to exon 5 (lack of exon 4 and 4b) and is comprised in spliceforms 2 and 4.
EKIRKALPNSEDLVKLAPDFDKIVESLSLLKDFFTSGSPEETAFRATDRGSESDKHFR K (SEQ ID NO: 33) corresponds to the amino acid sequences from exon 4 to exon 5 (lack of exon 4b) and is comprised in spliceforms 1 and 7.
GSPEETAFRATDRGSESDKHFRKVSDKEKIDQLQEELLHTQLKYQRILERLEKENKE LRK (SEQ ID NO: 34) corresponds to the amino acid sequences from exon 5 to exon 6 (lack of exon 5b) and is comprised in spliceforms 1, 2, 3 and 5.

Other amino acid sequences specific for a certain OPA1 spliceform can be derived from the amino acid sequences of the OPA1 spliceforms given herein below (e. g. Fig. 1).
Since an OPA1 isoform to be employed in context of the present invention may be derive from one particular OPA1 spliceform, the above mentioned amino acid sequences defining the different OPA1 spliceforms may also be used to determine the ratio of given OPAL1 isoforms as defined herein. For example, since the present invention provides evidence that OPA1-L1 be derived from spliceform 7 and OPA1-L2 be derived from spliceform 1, OPA1-L1 may, e. g. be characterized in that it comprises the amino acid sequence EKIRKALPNSEDLVKLAPDFDKIVESLSLLKDFFTSGSPEETAFRATD RGSESDKHFRK (SEQ ID NO: 33) and in that it not comprises the amino acid sequence GSPEETAFRATDRGSESDKHFRKVSDKEKIDQLQEELLHTQLKYQRILER LEKENKELRK (SEQ ID NO: 34) and OPA1-L2 may, e. g. be characterized in that it comprises the amino acid sequence EKIRKALPNSEDLVKLAPDFDKIVESLSLLKDFF TSGSPEETAFRATDRGSESDKHFRK (SEQ ID NO: 33) and GSPEETAFRATDRGS ESDKHFRKVSDKEKIDQLQEELLHTQLKYQRILERLEKENKELRK (SEQ ID NO: 34). However, since the OPA1 isoforms to be employed in context of the present invention may derive from the OPA1 splicefroms by (proteolytical) processing, not the complete amino acid sequences as given above, but fragments or derivatives thereof, may be used to determine a certain OPA1 isoform.
Particularly useful in context of the present invention, and exemplified in the appended examples (e. g. Fig.2), are SDS-PAGE-gels having a polyacrylamide concentration of 10%. However, it is also envisaged that SDS-PAGE-gels to be employed in context of the present invention have other polyacrylamide concentrations. E. g. said concentrations may be 1%, 2%, 3%, 4%, 5% or 10 % higher or lower than that of a 10% SDS-PAGE, e.g. than that of the 10% SDS-PAGE-gel as exemplified herein, but also other concentrations are envisaged. A person skilled in the art is readily in the position to transfer molecular weigth values deduced on the basis of an SDS-PAGE-gel having a certain polyacrylamide concentration, e. g. 10% as exemplified herein (e. g. Fig.2), to molecular weigth values deduced on the basis of an SDS-PAGE-gel having a different polyacrylamide concentration. As mentioned above, also other means than direct determination of a given molecular weight from an SDS-PAGE-gel may be used in context of this invention. For example, the appended experimental data provide for corresponding Western-blots.
The meaning of the term "Mass spectrometry" (MS) is and corresponding methods are known in the art. Particularly useful "mass spectrometry" methods to be employed in context of the present invention, and as exemplified herein (Example 3), are MALDI-MS or LC-MS/MS. Further "mass spectrometry" methods are known in the art and can easily be adapted to the specific needs of the present invention by a person skilled in the art.
The term "molecular weights being determined by mass spectrometry" means that the apparent molecular weight of a certain OPA1 isoform is determined by performing mass spectrometry analysis on said OPA1 isoform (e. g. as in example 3) and using the results of said mass spectrometry analysis to calculate said apparent molecular weight of said certain OPA1 isoform on the basis of the amino acid sequence of OPA1. Since eight alternative spliceforms exist of OPA1, having different amino acid sequences, the result of said calculation may vary, dependent on the spliceform, the amino acid sequence of which is used for said calculation. Examples of such determination of molecular weights by mass spectrometry are given herein (example 3). The principle of such determination is described in the following:
First, the amino acid sequences of certain peptide streches comprised in an OPA1 isoform to be analysed is determined by mass spectrometry (e. g. as in example 3). Second, the resulting amino acid sequences of said peptide streches are then compared with the amino acid sequences of the eight spliceforms of OPA1. Then, it is determined which certain OPA1 spliceforms comprise the amino acid sequences of said peptide streches and which do not. Subsequently, of the spliceforms that comprise the amino acid sequences of said peptide streches, that amino acid sequence it is estimated, which starts with the amino acid sequence of the most N-terminal peptide determined. Last, from said estimated amino acid sequence, the theoretical molecular weight of the respective OPA1 isoform to be analysed is calculated based on the known molecular weights of the amino acid residues comprised.
It is of note that the so determined theoretical molecular weight may be further increased by the presence of a few further N-terminally located amino acid residues present in the (proteolytically) processed mature OPA1 isoform. The person skilled in the art is readily in the position to determine said slightly increased molecular weight, by taking advantage of the teaching of the present invention. As a non limiting example, the determination of such slightly increased theoretical molecular weight of certain OPA1 isoforms is provided in the so called "refined analysis" of appended example 3
In context of the present invention, the large isoforms of OPA1 may comprise two isoforms (e. g. OPA1-L1 and OPA1-L2) and the small isoforms of OPA1 may comprise three isoforms (e. g. OPA1-S3, OPA1-S4 and OPA1-S5). However, it is also envisaged that further, possibly existing isoforms may assigned as large or small isoforms in context of the present invention. For instance, it is evident for a skilled person that, e. g., single bands of an SDS-PAGE/Western-blot as exemplified herein, may represent not only one, but several different isoforms and/or that further isoforms, larger or smaller than the particular isoforms defined herein may be present. For example, particularly the band corresponding to OPA1-S4 as defined herein may correspond to (a) further OPA1 isoform(s). Again, the gist of the present invention is based on the fact the determination of "small" versus "large" isoforms is illustrative for mitochondrial dysfunction and corresponding related disorders/diseases. Therefore, further, possibly existing isoforms may, e. g., be detectable by alternative comparable methods known in the art and may also be taken into consideration in the herein provided methods and means. For example, such methods may be SDS-PAGEs taking advantage of gels having very low polyacrylamide concentrations (e.g. 1%, 2%, 3% or 4%) and/or Western-blots taking advantage of radionuclide labelling, e. g. radionucleotide labelling of (secondary) antibodies used in said Western-blots, or other labelling approaches known in the art, e. g. other very sensitive labelling approaches being suitable for the detection of proteins being present in low amount(s)/concentration(s). Moreover, such methods may be a two dimensional gelelectrophoresis methods. These and orther alternative methods for detecting isoforms of certain proteins/genes, like OPA1, are known in the art. It is envisaged that such alternative methods may also be employed in context of the present invention.
However, it is preferred that each single band as evident from the SDS-PAGE analysis as employed and exemplified herein represents one single OPA1 isoform. Accordingly, in one embodiment of the present invention, the two large OPA1 isoforms as defined herein (e. g. OPA1-L1 and OPA1-L2) are represented by two single bands, and the three small OPA1 isoforms as defined herein (e. g. OPA1-S3, OPA1-S4 and OPA1-S5) are represented by three single bands occurring in an SDS-PAGE, e. g. an SDS-PAGE as exemplified herein.
In context of the present invention, the two large OPA1 isoforms are indicated by numbers 1 and 2, namely 1 for the largest and 2 for the second largest OPA1 isoform. The three small OPA1 isoforms are indicated by numbers 3, 4 and 5, namely 3 for the largest of the three small isoforms, 4 for the second largest of the three small isoforms and 5 for the smallest isoform. The numbering of the OPA1 isoforms to be employed in context of the present invention is also given in the appended examples and corresponding figures, e. g. Fig.2. In accordance thereto the OPA1 isoforms as employed in context of the present invention are termed as follows: OPA1-L/I1, L/I-OPA1#1, OPA1#1 or L/I1-OPA1 for the largest OPA1 isoform. OPA1-L/I2, L/I-OPA1#2, OPA1#2 or L/I2-OPA1 for the second largest OPA1 isoform. Large isoform(s) in context of the present invention is (are), e. g., OPA1-L1 and/or OPA1-L2. OPA1-S/s3, S/s-OPA1#3, OPA1#3 or S/s3-OPA1 for the largest of the three small OPA1 isoforms. OPA1-S/s4, S/s-OPA1#4, OPA1#4 or S/s4-OPA1 for the second largest of the three small isoforms. OPA1-S/s5, S/s-OPA1#5, OPA1#5 or S/s5-OPA1 for the smallest OPA1 isoform. Accordingly, small isoform(s) in context of the present invention is (are), e. g., OPA1-S3, OPA1-S4 and/or OPA1-S5.
It is of note that the specific numbering is indicative for the specific OPA1 isoform, and that the additional terming, like "I" for large; "s" for small or "OPA", "OPA1" or "OPA1#" for OPA1 may slightly vary. However the abbreviations "L" or "I" indicate large isoforms and "S" or "s" indicate small isoforms of OPA1.
In view of the teaching provided herein, also in the appended examples, the OPA1 isoforms employed in context of the present invention are defined as follows:
In one aspect of the present invention, the term "OPA1 isoform" means a protein encoded by the OPA1 gene, but particularly be derived from at least one of the different spliceforms of OPA1 (e. g. from at least one of spliceforms 1 to 8 as depicted in Figure 1), e. g. by posttranslational (e. g. proteolytical) processing, wherein said proteins are distinguishable by their molecular weight and/or (a) certain amino acid sequence(s). From the above, it is, inter alia, evident that an "OPA1 isoform" as employed in context of the present invention comprises (an) amino acid streche(s) which unambiguously characterize it as a polypeptide/protein derived from OPA1. In this context, "derived from OPA1" particularly means encoded by the OPA1 gene and/or generated from OPA1 by the herein described and defined OPA1 processing. Thus, an "OPA1 isoform" as employed can particularly be characterized by (a) certain amino acid strech(es) of any one od SEQ ID No: 2, 4, 6, 8, 10, 12, 14 or 16 or by (a) certain amino acid strech(es) encoded by any one od SEQ ID No: 1, 3, 5, 7, 9, 11, 13 or 15.
In context of the present invention, the term "molecular weight" may, inter alia, refer to the apparent molecular weight. Said apparent molecular weight can be determined by methods known in the art. E. g., said apparent molecular weight can be determined by SDS-PAGE, and, accordingly, also from Westem-blots, or can be calculated from the amino acid sequence of OPA1, particularly from the amino acid sequence(s) of the corresponding spliceform(s) by taking advantage of mass spectrometry methods. Examles of the determination of the OPA1 isoforms by using these techniques are given in the appended examples (e. g. example 2/3/11; Fig.2/3).
As already mentioned above, in context of the present invention, certain given molecular weigth values are apparent molecular weigth values. It is envisaged, that the certain molecular weigth values given herein may slightly vary, e. g. with respect to the molecular weigth of the protein present in vivo. Said variation may by in the range of 5 kD, 4 kD, 3 kD, 2 kD, 1 kD, 0.5 kD, 0.4 kD, 0.3 kD, 0.2 kD or 0.1 kD, whereby the smaller variations are preferred over the larger variations.
In context of the present invention, large isoforms comprise an isoform having an apparent molecular weight of about 97 kD (96.8 kD) (defined as OPA1-L1) or an isoform having an apparent molecular weight of about 92 kD (92.3 kD) (defined as OPA1-L2), said molecular weights being determined by SDS-PAGE analysis. Moreover, in context of the present invention large isoforms comprise an isoform having an apparent molecular weight of about 104 kD (104.0 kD) or, preferably, of about 105 kD (105.1 kD) (defined as OPA1-L1) or an isoform having an apparent molecular weight of about 99 kD (99.2 kD) or, preferably, of about 100 kD (100.0 kD) (defined as OPA1-L2), said molecular weights being determined by mass spectrometry.
The molecular weight values determined by mass spectrometry of OPA1-L1 and OPA1-L2 are given as averaged values of the four smallest (OPA1-L2) and the four largest (OPA1-L1) molecular weight values of the second column ("I-OPA1#1/#2") of the corresponding table in example 3.
In this context, it is of note that the present invention provides evidence that OPA1-L1 be derived from spliceform 7 and OPA1-L2 be derived from spliceform 1 or spliceform 4. Accordingly, it is particularly envisaged herein that OPA1-L has an apparent molecular weight determined by mass spectrometry of about 105 kD (104.9 kD) or, preferably, of about 106 kD (105.8 kD). OPA1-L2 is particularly envisaged to have an apparent molecular weight determined by mass spectrometry of about 101 kD (100.7 kD) or, preferably, of about 102 kD (101.5 kD), if determined on the basis of spliceform 1. If determined on the basis of spliceform 4, OPA1-L2 is particularly envisaged to have an apparent molecular weight determined by mass spectrometry of about 101 kD (100.8 kD) or, preferably, of about 102 kD (101.7 kD) (see second column ("I-OPA1#11#2") of the tables in example 3).
In addition to the above, the large OPA1 isoforms to be scrutinized in context of the present invention, namely OPA1-L1 and OPA1-L2, are characterized by comprising, e. g. as most N-terminal peptides, amino acid stretches or amino acid peptides comprising one or more of the following sequences: YLILGSAVGGGYTAK (SEQ ID No: 17), TFDQWK (SEQ ID No: 18), DMIPDLSEYK (SEQ ID No: 19), WIVPDIVWEIDEYIDFEK (SEQ ID No: 20), LAPDFDK (SEQ ID No: 21), IVESLSLLK (SEQ ID No: 22), ALPNSEDLVK (SEQ ID No: 23), DFFTSGSPEETAFR (SEQ ID No: 24) and TRLLKLRYLILGS (SEQ ID No: 25) and FWPARLATRLLKLRYLILGS (SEQ ID NO: 111).
It is evident from the teaching provided herein that the large OPA1 isoforms to be scrutinized in context of the present invention, namely OPA1-L and OPA1-L2, may (further) be characterized by further amino acid stretches or amino acid peptides of OPA1, or of particular OPA1 spliceforms, e. g. by further amino acid stretches or amino acid peptides of OPA1, or of particular the OPA1 spliceforms, lying more C-terminal to the above mentioned amino acid stretches or amino acid peptides. For instance, said further amino acid stretches or amino acid peptides of OPA1 may, in addition to or instead of the above mentioned sequences, comprise one or more of the following sequences: GLLGELILLQQQIQEHEEEAR (SEQ ID No: 26), AAGQYSTSYAQQK (SEQ ID No: 27) or IDQLQEELLHTQLK (SEQ ID No: 28)_.
However, it is of note that particularly OPA1-L2 does not comprise amino acid stretches or amino acid peptides comprising one or more of the following sequences: GLLGELILLQQQIQEHEEEAR (SEQ ID No: 26) or AAGQYSTSYAQQK (SEQ ID No: 27).
It is further evident from the teaching provided herein that, the large OPA1 isoforms to be scrutinized in context of the present invention, namely OPA1-L1 and OPA1-L2, may also be characterized by not comprising amino acid stretches or amino acid peptides comprising amino acid streches of OPA1, particularly of the OPA1 spliceforms, lying more N-terminal to those amino acid peptides comprising amino acid streches of OPA1, particularly of OPA1 spliceforms, that correspond to the most N-terminal peptides of the large OPA1 isoforms as defined herein, e. g. the peptide TRLLKLRYLILGS (SEQ ID No: 25) (see, e. g. Example 3; Fig. 1; Fig. 3).
Accordingly, the the large OPA1 isoforms to be scrutinized in context of the present invention, namely OPA1-L1 and OPA1-L2, are characterized by the feature that their apparent N-terminus correlates to amino acid position 102, preferably to amino acid position 95 of spliceforms 1 to 8 (SEQ ID No: 16, 14, 12, 10, 8, 6, 4 and 2, respectively).
As mentioned above, evidence is provided herein, that the OPA1 isoform OPA1-L1 be derived from spficeform 7 (SEQ ID Nos: 3/4) and the OPA1 isoform OPA1-L2 be derived from spliceform 1 (SEQ ID Nos: 15/16) or spliceform 4 (SEQ ID Nos: 9/10) of OPA1.
In context of the present invention, small isoforms comprise an isoform having an apparent molecular weight of about 88 kD (88.1 kD) (defined as OPA1-S3), an isoform having an apparent molecular weight of about 84 kD (84.4 kD) (defined as OPA1-S4) or an isoform having an apparent molecular weight of about 81 kD (80.9 kD) (defined as OPA1-S5), said molecular weights being determined by SDS-PAGE analysis. Moreover, in context of the present invention small isoforms comprise an isoform having an apparent molecular weight of about 92 kD (91.8 kD) or, preferably, of about 96 kD (95.9 kD) (defined as OPA1-S3), an isoform having an apparent molecular weight of about 89 kD (89.2 kD) or, preferably, of about 92 kD (91.8 kD) (defined as OPA1-S4) or an isoform having an apparent molecular weight of about 87 kD (86.8 kD) or, preferably, of about 87 kD (86.8 kD) (defined as OPA1-S5), said molecular weights being determined by mass spectrometry.
Based on the first estimate of appended example 3, the following applies with respect to the small OPA1 isoform employed herein:
OPA1-S3 is additionally characterized by comprising, e. g. as most N-terminal peptides, amino acid stretches or amino acid peptides comprising one or more of the following sequences: GLLGELILLQQQIQEHEEEAR (SEQ ID No: 26), AAGQYSTSYAQQK (SEQ ID No: 27) or IDQLQEELLHTQLK (SEQ ID No: 28).
OPA1-S4 is characterized by comprising, e. g. as most N-terminal peptides, amino acid stretches or amino acid peptides comprising one or more of the following sequences: AAGQYSTSYAQQK (SEQ ID No: 27) or IDQLQEELLHTQLK (SEQ ID No: 28) OPA1-S5 is characterized by comprising, e. g. as most N-terminal peptides, amino acid stretches or amino acid peptides comprising the following sequence: IDQLQEELLHTQLK (SEQ ID No: 28)
Moreover, the small OPA1 isoforms to be scrutinized in context of this invention, namely OPA1-S3, OPA1-S4 and OPA1-S5 are characterized by not comprising amino acid stretches or amino acid peptides comprising one or more of the following sequences: YLILGSAVGGGYTAK (SEQ ID No: 17), TFDQWK (SEQ ID No: 18), DMIPDLSEYK (SEQ ID No: 19), WIVPDIVWEIDEYIDFEK (SEQ ID No: 20), LAPDFDK (SEQ ID No: 21), IVESLSLLK (SEQ ID No: 22), ALPNSEDLVK (SEQ ID No: 23), DFFTSGSPEETAFR (SEQ ID No: 24) and TRLLKLRYLILGS (SEQ ID No: 25) It is of note that the small OPA1 isoforms to be scrutinized in context of this invention, namely OPA1-S3, OPA1-S4 and OPA1-S5 are further characterized by not comprising amino acid stretches or amino acid peptides of OPA1, particularly of one of the OPA1 spliceforms, lying more N-terminal to those amino acid peptides comprising amino acid streches of OPA1, particularly of OPA1 spliceforms, that correspond to the most N-terminal peptides of the small OPA1 isoforms as defined herein (see, e. g. Example 3; Fig. 1; Fig. 3).
Furthermore, OPA1-S4 is characterized by not comprising amino acid stretches or amino acid peptides comprising the following sequence: GLLGELILLQQQIQEHEEEAR (SEQ ID No: 26) and OPA1-S5 is characterized by not comprising amino acid stretches or amino acid peptides comprising one or more of the following sequences: GLLGELILLQQQIQEHEEEAR (SEQ ID No: 26) and AAGQYSTSYAQQK (SEQ ID No: 27).
Accordingly, OPA1-S3 is characterized by the feature that its apparent N-terminus correlates to amino acid position 227 of spliceform 8 (SEQ ID No: 2), to amino acid position 209 of spliceform 7 (SEQ ID No: 4), to amino acid position 191 of spliceform 6 (SEQ ID No: 6) or to amino acid position 173 of spliceform 4 (SEQ ID No: 10). Furthermore, OPA1-S4 is characterized by the feature that its apparent N-terminus correlates to amino acid position 249 of spliceform 8 (SEQ ID No: 2), to amino acid position 231 of spliceform 7 (SEQ ID No: 4), to amino acid position 213 of spliceform 6 (SEQ ID No: 6) or to amino acid position 195 of spliceform 4 (SEQ ID No: 10). Moreover, OPA1-S5 is characterized by the feature that its apparent N-terminus correlates to amino acid position 270 of spliceform 8 (SEQ ID No: 2), to amino acid position 252 of spliceform 7 (SEQ ID No: 4), to amino acid position 234 of spliceform 6 (SEQ ID No: 6), to amino acid position 233 of spliceform 5 (SEQ ID No: 8), to amino acid position 216 of spliceform 4 (SEQ ID No: 10), to amino acid position 197 of spliceform 3 (SEQ ID No: 12), to amino acid position 179 of spliceform 2 (SEQ ID No: 14) or to amino acid position 215 of spticeform 1 (SEQ ID No: 16).
Based on the refined analysis of appended example 3, the following preferred matter applies with respect to the small OPA1 isoform employed herein:
OPA1-S3 is additionally characterized by comprising, e. g. as most N-terminal peptides, amino acid stretches or amino acid peptides comprising one or more of the following sequences: IVESLSLLK (SEQ ID No: 22), DFFTSGSPEETAFR (SEQ ID No: 24), GLLGELILLQQQIQEHEEEAR (SEQ ID No: 26), AAGQYSTSYAQQK (SEQ ID No: 27) or IDQLQEELLHTQLK (SEQ ID No: 28). OPA1-S4 is characterized by comprising, e. g. as most N-terminal peptides, amino acid stretches or amino acid peptides comprising one or more of the following sequences: GLLGELILLQQQIQEHEEEAR (SEQ ID No: 26), AAGQYSTSYAQQK (SEQ ID No: 27) or IDQLQEELLHTQLK (SEQ ID No: 28). OPA1-S5 is characterized by comprising, e. g. as most N-terminal peptides, amino acid stretches or amino acid peptides comprising the following sequence: IDQLQEELLHTQLK (SEQ ID No: 28).
Moreover, the small OPA1 isoforms to be scrutinized in context of this invention, namely OPA1-S3, OPA1-S4 and OPA1-S5, are characterized by not comprising amino acid stretches or amino acid peptides comprising one or more of the following sequences: YLILGSAVGGGYTAK (SEQ ID No: 17), TFDQWK (SEQ ID No: 18), DMIPDLSEYK (SEQ 10 No: 19), WIVPDIVWEIDEYIDFEK (SEQ ID No: 20), LAPDFDK (SEQ ID No: 21), ALPNSEDLVK (SEQ ID No: 23) and TRLLKLRYLILGS (SEQ ID No: 25). It is of note that the small OPA1 isoforms to be scrutinized in context of this invention, namely OPA1-S3, OPA1-S4 and OPA1-S5, are further characterized by not comprising amino acid stretches or amino acid peptides of OPA1, particularly of one of the OPA1 spliceforms, lying more N-terminal to those amino acid peptides comprising amino acid streches of OPA1, particularly of OPA1 spliceforms, that correspond to the most N-terminal peptides of the small OPA1 isoforms as defined herein (see, e. g. Example 3; Fig. 1; Fig. 3). Furthermore, OPA1-S5 is characterized by not comprising amino acid stretches or amino acid peptides comprising one or more of the following sequences: GLLGELILLQQQIQEHEEEAR (SEQ ID No: 26) and AAGQYSTSYAQQK (SEQ ID No: 27).
Accordingly, OPA1-S3 is, inter alia, characterized by the feature that its apparent N-terminus correlates to amino acid position 172 of spliceform 7 (SEQ ID No: 4.
Furthermore, OPA1-S4 is characterized by the feature that its apparent N-terminus correlates to amino acid position 227 of spliceform 8 (SEQ ID No: 2), to amino acid position 209 of spticeform 7 (SEQ ID No: 4), to amino acid position 191 of spticeform 6 (SEQ ID No: 6) or to amino acid position 173 of spliceform 4 (SEQ ID No: 10).
Moreover, OPA1-S5 is characterized by the feature that its apparent N-terminus correlates to amino acid position 270 of spliceform 8 (SEQ ID No: 2), to amino acid position 252 of spliceform 7 (SEQ ID No: 4), to amino acid position 234 of spliceform 6 (SEQ ID No: 6), to amino acid position 233 of spliceform 5 (SEQ ID No: 8), to amino acid position 216 of spliceform 4 (SEQ ID No: 10), to amino acid position 197 of spticeform 3 (SEQ ID No: 12), to amino acid position 179 of spliceform 2 (SEQ ID No: 14) or to amino acid position 215 of spliceform 1 (SEQ ID No: 16).
It is of note that the molecular weight values of the OPA1 isoforms scrutinized herein which were determined by SDS-PAGE analysis, are given as averaged values corresponding to the molecular weight values of three different isoform bands within an SDS-PAGE/Westem-blot (see Fig.2).
In context of the present invention it is to be understood that the term "most N-terminal peptide(s)" means (a) peptide(s), e. g. (a) peptide(s) as defined above, that lies in the N-terminal region of the corresponding OPA1 isoform. Preferably, said peptide(s) is (are) the indeed most N-terminal peptide(s), what means that, e. g. when taking advantage of a particular protease during mass spectrometry analysis (e. g. see example 3), no other peptide can be found in the corresponding OPA1 isoform lying in a more N-terminal direction of said peptide. However, it is of note that the N-terminal amino acid(s) of said "most N-terminal peptide(s)" may not be the most N-terminal amino acid(s) of the corresponding OPA1 isoform(s). The most N-terminal amino acid(s) of the corresponding OPA1 isoform(s) may also correspond to a slightly more N-terminal amino acid position of OPA1, (particularly of the OPA1 spliceforms. For instance, said slightly more N-tenninal amino acid position may be a position lying 1 to 15, preferably 1 to 10, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 1 to 2 or 1 amino acid position(s) in the N-terminal direction of the amino acid sequence(s) corresponding to the "most N-terminal peptide(s)", e. g. the peptides as defined above.
Since, without being bound by theory, the particular amino acid sequence(s) of the "most N-terminal peptide(s)" as defined herein, e. g., depend(s) on the protease to be employed during mass spectrometry analysis (e. g. see example 3), the amino acid sequence(s) of the "most N-terminal peptide(s)" may slightly vary, e. g. in the above defined ranges in the N-terminal direction (and/or C-terminal direction). The person skilled in the art is readily in the position to figure out said slightly varied amino acid sequence(s) of the "most N-terminal peptide(s)", and accordingly the N-terminal amino acid of the OPA1 isoforms, by taking advantage of the teaching of the present invention, and, e. g., by applying mass spectrometry analysis taking advantage of different proteases, which, e. g. may cut the amino acid strand at different positions. Such mass spectrometry analysis are also envisaged to be employed in context of the present invention.
In view of the above, it is also of note that the amino acid positions of the different OPA1 spliceforms that correlates to the apparent N-termini of the corresponding OPA1 isoforms given herein above, may slightly vary. For instance, this variation may in the range of 1 to 15 amino acids in the N-terminal direction, wherein the smaller variations within this range are preferred.
The term "derivatives" or "derivatives thereof' as well as "homologous" as defined herein above, also apply, mutatis mutandis, in context of the peptides shown above, e. g. the peptides comprised in the OPA1 isoforms or the peptides that characterize the OPA1 spliceforms. Moreover, the term "derivatives" or "derivatives thereof" also refers to (a) fragment(s), e. g. (a) fragment(s) of the peptides shown above, e. g. the peptides comprised in the OPA1 isoforms or the peptides that characterize the OPA1 spliceforms. The term "fragment(s)" means amino acid streches of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 50, 100 or 150 amino acids. Also amino acid streches having other numbers of amino acids are envisaged.
In terms of the present invention the term "derivatives" or "derivatives thereof" also comprises homologies as well as conservative amino acid exchanges and further known modifications.
The term "determination of the identity, amount and/or ratio of large and/or small isoforms of the optic atrophy 1 protein (OPA1)" also refers to the determination of the identity, amount and/or ratio of N-terminal fragments being derived from posttranslational (e. g. proteolytical) processing of (an) OPA1 isoform(s)/spliceform(s) as defined herein, particularly of (an) large OPA1 isoform(s) as defined herein. Moreover, it is envisaged that further OPA1 fragments, e. g. C-terminal fragments of OPA1, being, without bound by theory, derived from posttranslational (e. g. proteolytical) processing of (an) OPA1 isoform(s)/spliceform(s) as defined herein, are scrutinized in context of the present invention, in order to diagnose mitochondrial disorders/dysfunctions, in particular the susceptibility for, predisposition for or the presence of a disorder correlated with mitochondrial dysfunction or mitochondrial disease.
Without being bound by theory, the (prolonged) posttranslational (e. g. proteolytical) processing of (an) OPA1 isoform(s)/spliceform(s) may result in short C-terminal fragments of OPA1, e. g. C-terminal fragments of OPA1 shorter than OPA1-S5 as defined herein. Again without being bound by theory, (an accumulation of) said short C-terminal fragments of OPA1 may be diagnostic for mitochondrial disorders/dysfunctions, in particular the susceptibility for, predisposition for or the presence of a disorder correlated with mitochondrial dysfunction or mitochondrial disease. Accordingly the determination of the identity, amount and/or ratio of such short C-terminal fragments of OPA1 is also disclosed herein, in order to diagnose mitochondrial disorders/dysfunctions, in particular the susceptibility for, predisposition for or the presence of a disorder correlated with mitochondrial dysfunction or mitochondrial disease.
The person skilled in the art is readily in a position to adapt the teaching provided herein to corresponding methods, kits and uses taking advantage of scrutinizing the above mentioned N-terminal and/or short C-terminal fragments of OPA1.
The term "determination of the identity, amount and/or ratio of large and/or small isoforms of the optic atrophy 1 protein (OPA1)" refers to the determination of the identity, amount and/or ratio of OPA1 isoforms via detection of any amino acid strech lying between the N-terminal amino acid of the "most N-terminal peptide(s)" defined herein of the OPA1 isoform(s) to be scrutinized, alternatively and preferred between the N-terminal amino acid of the OPA1 isoform to be scrutinized and the C-terminus of OPA1, particularly of the OPA1 spliceforms. Said C-terminus of OPA1, particularly of the OPA1 spliceforms, without being bound by theory, also corresponds to the C-terminus of the OPA1 isoforms defined herein.
For instance, said amino acid strech may be any epitope-bearing portion, or, e. g. any other portion to which a binding molecule as defined herein can bind, lying between the N-terminal amino acid of the "most N-terminal peptide(s)" defined herein of the OPA1 isoform(s) to be scrutinized, alternatively and preferred between the N-terminal amino acid of the OPA1 isoform to be scrutinized and the C-terminus of OPA1, particularly of the OPA1 spliceforms. For instance, said detection may be a detection method as defined and exemplified herein, e. g. a detection method taking advantage of the antibodies as defined and exemplified herein, or a detection method taking advantage of other binding molecules as defined herein.
In a non-limiting example, it is envisaged in context of the present invention that the ratio of the large OPA1 isoforms as defined herein, namely OPA1-L1 and OPA1-L2, can be determined via specific detection of any amino acid strech of the large OPA1 isoforms lying in N-terminal direction to the amino acid streches corresponding to the N-terminal amino acids of the "most N-terminal peptide(s)" defined herein of the small OPA1 isoform(s), alternatively and preferred lying in N-terminal direction to the amino acid streches corresponding to the N-terminal amino acid of the small OPA1 isoforms. In analogy to the above, said amino acid strech to be detected may be any epitope-bearing portion, or, e. g. any other portion to which a binding molecule as defined herein can bind and said detection may be a detection method as defined and exemplified herein, e. g. a detection method taking advantage of the antibodies as defined and exemplified herein, or a detection method taking advantage of other binding molecules as defined herein.
In another particular embodiments of this invention, it is envisaged to diagnostically distinguish between various types of mitochondrial dysfunction(s)/disease(s). In particular, it is envisaged to differentiate between mitochondrial dysfunction(s)/disease(s) dependent on depletion of mitochondrial DNA and other types of mitochondrial dysfunction(s)/disease(s). Moreover, a quantitative measure of mitochondrial dysfunction is also envisaged. These embodiments of this invention are also based on the findings provided herein and demonstrated in the appended examples (e.g. example 7), e. g. the findings that certain mitochondrial dysfunction(s)/disease(s) correlate with a particular pattern of large and/or small isoforms of the optic atrophy 1 protein (OPA1), e.g. with a certain ratio of OPA1 isoforms (e.g. a certain ratio of OPA1-S5 compared to all OPA1 isoforms (e.g. see example 7 and corresponding table)). The Person skilled in the art is, based on the teaching of the present invention, readily in the position to figure out (further) particular patterns or corresponding ratios of large and/or small isoforms of the optic atrophy 1 protein (OPA1) being specific for certain types of mitochondrial dysfunction(s)/disease(s).
In context of the present invention, it is intended that the ratio of large and/or small isoforms of OPA1 is determined by optical, spectrophotometric and/or densitometric measurements or analysis. Such determination methods are well known in the art. A particular choice of such methods is described in the appended examples. For instance, such methods comprise the SDS-PAGE analysis, Western blots, ELISA, RIA, CLIA, IRMA and/or EIA. These and further methods are known in the art and are, e. g., described in "Cell Biology: Laboratory manual 3rd edition" (2005, J. Celis, editor. Academic Press, New York).
It is also intended that the identity, amount or ratio of large and/or small isoforms of OPA1 is determined by peptide analysis. Again, such peptide analysis methods are well known in the art. For example such peptide analysis methods comprise mass spectrometry methods, like MALDI-MS or LC-MS/MS. The use of these particular mass spectrometry methods are described in the appended examples.
A person skilled in the art is able to figure out further methods for the determination of the identity, amount or ratio of large and/or small isoforms of OPA1. For examples such methods are array-based protein detection and quantification (e. g. as described in Nettikadan, 2006, Mol Cell Proteomics. 5:895-901), immuno-PCR (IPCR; e. g. as described in Adler, 2005, Adv Clin Chem. 39:239-92; Case, 1997, Biochem Soc Trans. 25:374S; Guo, 2006, Nucleic Acids Res. 34:e62; Imai, 1996, Tanpakushitsu Kakusan Koso. 41:614-7; Ruzicka, 1993, Science. 260:698-9; Sano, 1992, Science. 258:120-2; Zhou, 1993, Nucleic Acids Res. 21:6038-9), fluorescent immuno-PCR (e. g. as described in Niemeyer, 1997, Anal Biochem. 246:140-5), Immuno-detection amplified by T7 RNA polymerase (IDAT; e. g. as described in Zhang, 2001, Proc Natl Acad Sci U S A. 98:5497-502), and quantitative mass spectrometry (e.g. as described in Fomer, 2006, Mol Cell Proteomics, 5, 608-19; Zhou, 2004, Methods Mol Biol, 261,511-8).
It is envisaged in context of the present invention that the methods provided are carried out on (a) biological or medical sample(s). Said biological or medical sample(s) may comprise cell(s) or tissue(s). For example, said biological or medical sample(s) may comprise biological material of biopsies. The meaning of "biopsies" is known in the art. For instance, biopsies comprise cell(s) or tissue(s) taken, e. g. by the attending physician, from a patient, suffering from the herein defined mitochondrial disease(s), dysfunction(s) or disorder(s) or showing the herein defined clinical and/or pathological symptoms.
The method as described herein is to be carried out on biological/medical samples which comprise mitochondria, i.e. on tissues or cells or on biological fluids which comprise some cells comprising mitochondria. Yet, it is also envisaged that the method of the present invention is carried out on biological samples which do not comprise complete cells but cellular fragments or mitochondrial fragments. Accordingly, the method of the present invention is not limited to biological/medical samples which comprise or are cells or tissues.
As documented in the appended examples, in one embodiment, muscles cells are to be investigated in context of this invention, i.e. the non-limiting results provided herein document the usefulness of the present invention in particular in context of muscular disorders, like MERRF. As mentioned above, MERRF is myoclonic epilepsy with ragged-red fiber syndrome and MERRF patients suffering from severe myopathy. It was demonstrated herein (e. g. example 5), that in a cybrid cell line derived from a MERRF patient, cells showed highly fragmented mitochondria compared to control cells (Fig.6ab). The fragmentation staus of the mitochondria correlates with the pattern of the five detected OPA1 isoforms in a manner that in the MERRF cells large OPA1 isoforms (particularly OPA1-L1 and OPA1-L2) are reduced and the small OPA1 isoforms (particularly the smallest isoform of OPA1, OPA1-S5) are enhanced.
Examplarily, but not limiting, the biological or medical sample to be analysed in context of the present invention is or is derived from blood, plasma, white blood cells, urine, semen, sputum, cerebrospinal fluid, lymph or lymphatic tissues or cells, muscle cells, heart cells, nerve cells, cells from spinal cord, brain cells, liver cells, kidney cells, cells from the intestinal tract, colon cells skin, bone, bone marrow, placenta, amniotic fluid, hair, hair and/or follicles, stem cells (embryonal, neuronal, and/or others), primary or immortalized cell lines (lymphocytes, macrophages, or any cell lines derived from any of the aforementioned tissues), tumor-derived cell lines such as HeLa cell lines, neuroblastoma cell lines or insulinomo cell lines. The the biological or medical sample as defined herein may also be or be derived from biopsies (see also herein below).
It is envisaged that the determination of the identity of large and/or small OPA1 isoforms comprises the step of determining the identity of at least one OPA1 isoform as defined herein.
The determination of the "identity" of at least one OPA1 isoform relates to the determination of the presence of at least one of the herein defined OPA1 isoforms (OPA1-L1, -L2, -S3, -S4 and/or -S5). At least the presence of one, of two, of three, of four or of all five isoforms as defined herein is determined. The presence of the corresponding isoform may be determined by methods known in the art and as described herein, e.g. by determining the corresponding band(s) on SDS-PAGE gels, by Westem-Blot analysis, by other immunological / immunobiochemical / immunochemical tests (like ELISA-tests, RIA-tests, etc), by peptide analysis (for example by mass spectrometry, etc) and the like.
It may, inter alia, be deduced whether in a given sample, the OPA1 large isoforms are present and/or in what amount they are present. Similarly, the determination of the presence of a given (or all) small OPA1 isoforms is useful. Again, also the quantitative (as well as the qualitative) identity of the individual OPA1 isoforms as described herein may be deduced and measured. As detailed herein, the gist of the present invention is based on the surprising finding that a determined reduction of large OPA1 isoforms as described herein (OPA1-L1 and/or -L2) and/or a determined increase of small OPA1 isoforms as described herein (OPA1-S3, -S4 and/or -S5, in particular OPA1-S5) is indicative for the presence of or the susceptibility to a mitochondrial disease/disorder/dysfunction.
It is further envisaged that the determination of the amount of large and/or small OPA1 isoforms comprises the step of determining the amount of at least one OPA1 isoform as defined herein. As mentioned above, also comprised in the methods provided herein are tests for the presence or the absence of individual OPA1 isoforms as defined above. The amount of each individual OPA1 isoform as defined herein (OPA1-L1, -L2, -S3, - S4, -S5) may be deduced by methods known in the art, like quantitative measurements on Westem-blots or the quantification of the amount by known techniques directly form SDS-PAGEs, like the determination of quantities in stained gels (like silver-stained, Coommassie-stained gels, and the like). Further quantitative methods comprise but are not limited to methods like ELISA-tests and the like. The methods for quantification are well known on the art, see e.g. Cell Biology: Laboratory manual 3rd edition. J. Celis, editor. Academic Press, New York.
In addition, the quantitative amount of at least one isoform as defined herein is determined. It is also envisaged that at least two isoforms, at least three, at least four or all five isoforms of OPA1 as defined herein are determined. As documented in the appended examples, it is preferred that at least one isoform of the two large isoforms and/or at least one isoform of the small isoforms is to be determined. Most preferably, if the small isoform is to be determined, at least the OPA1-S5 isoform is to be deduced.
As mentioned above, the gist of the present invention lies in the surprising finding that in pathological samples (or samples which tend to become pathological), the amount of large isoforms of OPA1 is reduced and the amount of small isoforms is enhanced in said samples. The corresponding determination of the individual amount of each individual isoform may comprise a comparison to the corresponding standard amount (for example obtained from samples form healthy volunteers) or in direct comparison to samples obtained from healthy individuals.
A mitochondrial disorder (as well as a susceptibility to the same) may be diagnosed by determining the amount of the individual OPA1 isoforms described herein. The presence or the susceptibility to a mitochondrial dysfunction can be determined by evaluating the amount of "large" versus "small" OPA1 isoforms as described herein, wherein a higher amount of small isoforms, in particular isoform S5 is indicative for a mitochondrial dysfunction/disorder or a susceptibility thereto. However, it is of note that not only the amount of "small" versus "large" (and accordingly the ratio between these OPA1 isoforms) may be measured in a given sample in context of this invention, but it is also envisaged that the amount of an individual isoform is measured and compared to a corresponding standard, for example a standard derived from or obtained from samples form healthy individuals, in particular healthy humans.
In accordance with the present invention, it is envisaged that also the ratio of individual OPA1 isoforms in comparison to the other OPA1 isoforms as described herein are determined. As detailed below and as exemplified in the appended examples, also said ratio of individual OPA1 isoforms to the other four, three, two or single OPA1 isoforms, or to all five OPA1 isoforms, as described herein are of diagnostic relevance. Again, this diagnostic relevance for the elucidation whether a mitochondrial disorder is present or whether an individual has a prevalence for a mitochondrial dysfunction/disesase is based on the surprising finding that such affected individuals or individuals susceptible to such an disorder/dysfunction/disease are characterized by displaying in his/her biological samples a comparatively higher amount of smaller OPA1 isoforms than large OPA1 isoforms as described herein. Accordingly, also the ratio between small to large (or also large to small) OPA1 isoforms is of diagnostic relevance. In particular, a higher amount of OPA1 isoform S5 in comparison to the other isoforms, in particular the large isoform(s) L1 and/or L2 is diagnostic for the presence or the prevalence of a mitochondrial disorder/dysfunction/disease. Yet, as documented herein, also a higher amount of the OPA1 isoform S3 is of high diagnostic value for the determination of such a disorder.
Exemplarily, but not limiting, ratios between individual or combined OPA1 isoforms being of diagnostic relevance are given in the following. Such ratios can also be deduced from the appended examples (e.g. example 7 and 8).
The ratio between OPA1-S5 and all OPA1 isoforms may vary between 1% and 25%, preferably between 5% and 15%, for healthy patients and between 10% and 75%, preferably between 15% and 60%, for affected patients. For example, said ratio may be 10% +/- 1, 2, 3, 4, or 5% (particularly 9.9%) for healthy patients and 25% +/- 1, 2, 3, 4 or 5% (particularly 24.9%) for affected patients. Accordingly, the increase of said ratio of affected patients compared to healthy ones may be in the range of 1 to 12 fold, more preferably in the range of 2 to 8 fold, more preferably in the range of 2 to 6 fold and more preferably in the range of 2 to 3 fold or 4 to 5 fold. For example, said increase may be 2.5 fold or 4.4 fold.
In view of the above, it is envisaged in context of the present invention that, e. g. a ratio between OPA1-S5 and all OPA1 isoforms of more than 10 %, preferably of more than 15%, more preferably of more than 20 % and most preferably of more than 25% is diagnostic for the "affected patients". It is to be understood that, in context of the present invention, "affected patients" are patients suffering from a susceptibility for, a predisposition for or the presence of a disorder correlated with mitochondrial dysfunction or mitochondrial disease.
The ratio between OPA1-S4 and all OPA1 isoforms may be 27% +/- 1, 2, 3, 4, or 5% (e. g. 27.2%) for healthy patients and 28% +/- 1, 2, 3, 4, or 5% (e. g. 28.2%) for affected patients. Accordingly, the increase of said ratio of affected patients compared to healthy ones may be in the range of 1 to 1.5 fold. As mentioned herein above, a decrease of said ratio of affected patients compared to healthy ones, particularly in context of OPA1-S4, is also possible. For example, it is also possible that said ratio does not change between affected patients and healthy ones. The ratio between OPA1-S3 and all OPA1 isoforms may be 10% +/- 1, 2, 3, 4, or 5% (e. g. 9.9%) for healthy patients and 19% +/- 1, 2, 3, 4, or 5% (e. g. 19.2%) for affected patients. Accordingly, the increase of said ratio of affected patients compared to healthy ones may be in the range of 1 to 5 fold, more preferably in the range of 1.5 to 3 fold and more preferably in the range of 1.8 to 2.3 fold. For example, said increase may be particularly 1.9 fold.
The ratio between OPA1-S3, -S4 and -S5 and all OPA1 isoforms may be 47% +/- 1, 2, 3, 4, 5 or 10% (e. g. 47.4%) for healthy patients and be 93% +/- 1, 2, 3, 4, 5 or 10% (e. g. 93.0%) for affected patients. Accordingly, the increase of said ratio of affected patients compared to healthy ones may be in the range of 1.5 to 3 fold, more preferably in the range of 1.8 to 2.5 fold and more preferably in the range of 1.9 to 2.2 fold. For example, said increase may be particularly 2.0 fold.
The ratio between OPA1-S3 and -S5 and all OPA1 isoforms may be 20% +/- 1, 2, 3, 4, or 5% (e. g. 20.2%) for healthy patients and between be 65% +/- 1, 2, 3, 4, 5 or 10% (e. g. 64.8%) for affected patients. Accordingly, the increase of said ratio of affected patients compared to healthy ones may be in the range of 2 to 5 fold, more preferably in the range of 2.5 to 4 fold and more preferably in the range of 3 to 3.6 fold. For example, said increase may be particularly 3.2 fold.
In context of values of the ratio of OPA1 isoforms, e.g. the values given above, it is to be understood that within the ranges given for the values, the smaller variations are preferred.
It is intended that the ratios to be determined in context of the present invention may also differ from the ones exemplified above. As already mentioned above, examples that, in a non limiting manner, describe the evaluation of such ratios are given herein below (e. g. Example 7 and 8).
Inter alia, in context of the present invention, the term "ratio" or "density ratio", inter alia, refers to a comparison of density values of bands corresponding to OPA1 isoforms, as, e. g., derived from an SDS-PAGE/Western-blot. Methods how such density values can be obtained are known in the art and exemplified in the appended non limiting examples.
It is to be understood that not only the comparison of small (OPA1-S3, -S4 and/or - S5) versus large isoforms of OPA1 (OPA1-L1 and/or -L2) or small or large versus other or all OPA1 isoforms derived from an individual patient sample or sample to be tested is of diagnostic relevance, but that also a comparison to an healthy control or a corresponding standard is of relevance and can, in accordance with the teachings provided herein, be obtained. This applies, mutatis mutantis, for all diagnostic methods provided herein.
As mentioned above, the term "determination of the ratio of OPA1 isoforms" is not limited to the determination of the ratio between one individual OPA1 isoform and one other individual OPA1 isoform as defined herein, but comprises also the determination of (a) ratio(s) between one individual isoform and at least two other OPA1 isoforms or even all other four OPA1 isoforms or even all five OPA1 isoforms as described herein. Accordingly, the determining the susceptibility for, predisposition for or the presence of a disorder correlated with mitochondrial dysfunction, also comprises a method wherein said determation of the ratio comprises

(a) the determination of the ratio of at least one large OPA1 isoform to at least one small OPA1 isoform;
(b) the determination of the ratio of at least one small OPA1 isoform to at least one large OPA1 isoform;
(c) the determination of the ratio of one OPA1 isoform to all five OPA1 isoforms or to all five OPA1 isoforms plus further OPA1 isoforms;
(d) the determination of the ratio of one OPA1 isoform to four OPA1 isoforms;
(e) the determination of the ratio of two OPA1 isoforms to three OPA1 isoforms,;
(f) the determination of the ratio of three OPA1 isoforms to two OPA1 isoforms; or
(g) the determination of the ratio of four OPA1 isoforms to one OPA1 isoforms;
(h) the determination of the ratio of all five OPA1 isoforms or of all five OPA1 isoforms plus further OPA1 isoforms to one OPA1 isoforms.

Moreover, all other possible combinations are envisaged in context of the present invention.
In an particular embodiment of the present invention, the ratio between at least one large OPA1 isoform and at least one small isoform is determined.
It is preferred in context of the present invention that the ratio between at least one small OPA1 isoform (e. g. OPA1-S5) and (all) five or more OPA1 isoforms, at least four OPA1 isoforms, at least three OPA1 isoforms, at least two OPA1 isoforms or at least one OPA1 isoforms is determined. It is more preferred in context of the present invention that the ratio between the OPA1-S5 as defined herein and at least one other OPA1 isoform as defined herein is determined.
It is most preferred in context of the present invention that the ratio between the OPA1-S5 as defined herein and all five OPA1 isoforms as defined herein is determined.
The person skilled in the art is readily in a position to determine the ratio of individual (or more) OPA1 isoforms as described herein by methods known in the art, like for example densitometric, spectrophotometric, luminescent, autoradiographic or fluorescent quantification methods. Also in this context, methods comprising tests with specific anti-OPA1 isoform antibodies (also specific antibodies against individual OPA1-isoforrns as provided herein) are useful. Accordingly, methods, like Westem-blot analysis or ELISA/RIA-tests may be employed to determine the OPA1 isoform ratio(s). Corresponding non-limiting examples are illustrated in the appended experimental part.
As discussed herein above, the methods as provided herein are based on the determination of small OPA1 isoforms (S3, S4 and/or S5) as provided herein versus large OPA1 isoforms (L1 and/or L2). However, it is to be understood that the methods provided herein also comprise an evaluation whether in a given sample the amount of large OPA1 isoforms (L1 and/or L2) is lower, and/or the amount of small OPA1 isoforms (S3, S4 and/or S5; preferably S3 and/or S5; more preferably S5) is higher in comparison to an individual healthy control sample, said sample being derived from either another (non-affected) tissue or cell of the individual to be diagnosed and/or assessed or said sample being a control sample from another healthy individual or a pool of samples from healthy individuals. It is also envisaged that the samples to be assessed are compared to (a) corresponding standard, either a standard which is obtained from affected individuals and/or healthy individuals. It is within the routine skills of the person skilled in the art to obtain corresponding standards.
In context of the present invention it is also envisaged that the determination of the ratio of large and/or small OPA1 isoforms comprises the step of determining the ratio between at least one OPA1 isoform as defined herein and an external standard. However, it is also envisaged that for all other methods provided herein, internal standards may be employed. Internal standard are known in the art. As a non limiting example, β-actinmay be employed as an internal standard in context of the present invention.
In further embodiments of the present invention, the herein described method for determining the susceptibility, predisposition or the presence of a disorder correlated with mitochondrial dysfunction or disease, comprising the in vitro determination of the ratio of large and small isoforms of OPA1 in a given sample comprises a comparison of the obtained data/values to data/values obtained from a healthy control/standard and/or a comparison of the obtained data/values to data/values obtained from a known diseased control/standard and/or a comparison of the obtained data/values to data/values obtained for an internal standard, e. g. for β-actin.
It is further envisaged that the methods provided herein comprise the steps of determing in a sample the ratio of at least two OPA1 isoforms selected from the group consisting of OPA1-L1, -L2, -S3, -
S4 and -S5, wherein said ratio of said isoforms is compared to the corresponding isoforms of a healthy control or normal standard.
Herein disclosed is a kit useful for the detection and/or diagnosis of a mitochondrial dysfunction, of a disorder correlated with mitochondrial dysfunction or of a mitochondrial disease, comprising an assay for determining the presence, the absence, the identity, the amount or ratio of at least one OPA1 isoform selected from the group consisting of OPA1-L1, -L2,- S3, -S4 and -S5, as defined herein .
Herein disclosed is a kit for the detection and/or diagnosis of a mitochondrial dysfunction, of a disorder correlated with mitochondrial dysfunction or of a mitochondrial disease, comprising a binding molecule which specifically binds to/interacts with optic atrophy 1 protein (OPA1) or a a binding molecule which specifically binds to/interacts with an isoform of optic atrophy 1 protein (OPA1) as defined herein
Additionally, uses of a binding molecule which specifically binds to/interacts with optic atrophy 1 protein (OPA1) or a binding molecule which specifically binds to/interacts with an isoform of optic atrophy 1 protein (OPA1) as defined herein for the preparation of a diagnostic composition for the detection of a disorder correlated with mitochondrial dysfunction or mitochondrial disease are described herein.
The binding molecule may be selected form the group consisting of antibodies, affybodies, trinectins, anticalins, aptamers, RNAs, PNAs and the like.
The person skilled in the art is readily the position to use and to obtain specific binding molecules, useful in the methods, kits, assays provided herein. These molecules are directed and bind specifically to or specifically label OPA1 and/or the isoforms of OPA1 provided and described herein. Non-limiting examples of these binding molecules may be selected from aptamers (Gold, Ann. Rev. Biochem. 64 (1995), 763-797)), aptazymes, RNAi, shRNA, RNAzymes, ribozymes (see e.g., EP-B1 0 291 533 , EP-A1 0 321 201 , EP-B1 0 360 257 ), antisense DNA, antisense oligonucleotides, antisense RNA, si RNA, antibodies (Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988), affibodies (Hansson, Immunotechnology 4 (1999), 237-252; Henning, Hum Gene Ther. 13 (2000), 1427-1439), trinectins (Phylos Inc., Lexington, Massachusetts, USA; Xu, Chem. Biol. 9 (2002), 933), anticalins, or the like. These compounds are, for example, described in EP 1 017 814 . Said European patent also describes the process of preparing such anticalins with the ability to bind a specific target.
The term "aptamer" means nucleic acid molecules that can bind to target molecules. Aptamers commonly comprise RNA, single stranded DNA, modified RNA or modified DNA molecules. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold (1995), Ann. ).
A preferred binding molecule is an antibody specific for OPA1 or a particular isoform of OPA1 as defined herein or a group of isoforms of OPA1 as defined herein. Such an antibody, e. g., may bind to the amino acid streches or amino acid peptides defined herein of OPA1 or of a particular isoform of OPA1 as defined herein or of a group of isoforms of OPA1 as defined herein. However, it is to be understood that such an antibody may also bind to other amino acid streches or amino acid peptides of OPA1 or of a particular isoform of OPA1 as defined herein or of a group of isoforms of OPA1 as defined herein also.
It is envisaged that such antibody (but also the other binding molecules ) is specific for one single isoform of OPA1, preferably for OPA-L1, -L2, -S3, -S4 or -S5, more preferably for OPA1-S5. For example, such antibody (or other binding molecules) specific for one single isoform of OPA1 are envisaged to be particularly employed to determine in a sample the presence, the absence, the identity or the amount of at one single OPA1 isoform selected from the group consisting of OPA1-L1, -L2,- S3, -S4 and -S5. Said presence, absence, identity or amount of said isoform may then be compared to the corresponding isoform of a healthy control or an internal or normal standard.
In this context, it is to be understood that the person skilled in the art is, based on the teaching provided herein, readily in a position to deduce (further) amino acid streches/peptides being specific for a particular isoform of OPA1 as defined herein or of a group of isoforms of OPA1 as defined herein. Moreover, the teaching provided herein enables the person skilled in the art to figure out the entire amino acid sequences of every single OPA1 isoform as defined herein. For example, this can be done by starting from the disclosure content of this invention and the corresponding appended examples and applying further peptide analysis methods, like mass spectrometry (e. g. MALDI-MS and/or LC-MS/MS).
As evident form the disclosure provided herein and in particular from the appended examples, the methods, kits and uses provided herein are particularly useful in the in vitro diagnoses of mitochondrial diseases/dysfunctions/disorders. Particularly useful are in this context antibodies, antibody molecules, antibody derivatives, which specifically interact with OPA1 and/or the isoforms of OPA1 as described herein. The term "antibody/antibodies" as employed herein also comprise antibody derivatives, antibody fragments and the like.
The antibody described herein can be, for example, polyclonal or monoclonal. The term "antibody" also comprises derivatives or fragments thereof which still retain the binding specificity. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. These antibodies can be used for the monitoring of the presence, absence, amount, identiy and/or ratio of OPA1 and/or the herein defined OPA1 isoforms, in particular in diagnosis. Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). Accordingly, also phage antibodies can be used.
Chimeric, single chain and humanized antibodies, as well as antibody fragments, like, inter alia, Fab fragments can also be used. Antibody fragments or derivatives further comprise F(ab')2, Fv or scFv fragments; see, for example, Harlow and Lane, loc. cit. Various procedures are known in the art and may be used for the production of such antibodies and/or fragments. Thus, the (antibody) derivatives can be produced by peptidomimetics. Further, techniques described for the production of single chain antibodies (see, inter alia, US Patent 4,946,778 ) can be adapted to produce single chain antibodies to polypeptide(s) as defined herein. Also, transgenic animals may be used to express humanized antibodies to OPA1 or OPA1 isoforms as described herein. Most preferably, the antibody to be employed is a monoclonal antibody. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique (Köhler and Milstein Nature 256 (1975), 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Techniques describing the production of single chain antibodies (e.g., US Patent 4,946,778 ) can be adapted to produce single chain antibodies to OPA1 or OPA1 isoforms. Accordingly,
the term "antibody molecule" relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules. Furthermore, the term relates, as discussed above, to modified and/or altered antibody molecules, like chimeric and humanized antibodies. The term also relates to monoclonal or polyclonal antibodies as well as to recombinantly or synthetically generated/synthesized antibodies. The term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab')2. The term "antibody molecule" also comprises bifunctional antibodies, trifunctional antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins.
The binding molecules provided herein are to be used in the methods, and kits as well as assays provided in the context of the evaluation of OPA1 and OPA1 isoforms, in particular in the assay for OPA1 proteolytic isoforms (like OPA1-S3, -S4, -S5, in particular OPA1-S3 and OPA1-S5 and most preferably OPA1-S5) in a given sample to be tested or analyzed.
In accordance with the present invention by the term "sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing polynucleotides or polypeptides or portions thereof. As indicated, biological samples include body fluids (such as blood, sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the polynucleotides of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. A biological sample which includes mitochondria, mitochondrial DNA, mitochondrial proteins, mitochondrial fragments (e. g. fragments of the mitochondrial outer and/or, more preferred, inner membranes) is preferred as a source.
The diagnostic composition optionally comprises suitable means for detection. Binding molecule or detection means described above are, for example, suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Examples of well-known carriers include glass, polystyrene, polyvinyl ion, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble . As documented in the examples, a partiuclar preferred solid phase may also be the membranes used in Western blots. It is evident that not only the "binding molecule" but also the Target molecule (OPA1, OPA1 fragments or OPA1 isoforms as described herein) may be bound to the "solid phase" and may be tested with the "binding molecules" provided in "liquid phase".
Solid phase carriers are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes. Suitable methods of immobilizing of binding molecules like, anticalins, antibody(ies), aptamer(s), polypeptide(s), etc. on solid phases include but are not limited to ionic, hydrophobic, covalent interactions or (chemical) crosslinking and the like. Examples of immunoassays are competitive and non-competitive immunoassays in either a direct or indirect format. Commonly used detection assays can comprise radioisotopic or non-radioisotopic methods. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Western blot assay. Furthermore, these detection methods comprise, inter alia, IRMA (Immune Radioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay). Furthermore, the diagnostic compounds may be are employed in techniques like FRET (Fluorescence Resonance Energy Transfer) assays.
Appropriate labels and methods for labeling are known to those of ordinary skill in the art. Examples of the types of labels which can be used, include inter alia, fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase, β-galactosidase, alkaline phosphatase), radioactive isotopes (like 32P, 33P, 35S or 125I), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums). A variety of techniques are available for labeling biomolecules, are well known to the person skilled in the art
and comprise, inter alia, covalent coupling of enzymes or biotinyl groups, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases). Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immunoassays", Burden and von Knippenburg (Eds), Volume 15 (1985); "Basic methods in molecular biology", Davis LG, Dibmer MD, Battey Elsevier (1990); Mayer, (Eds) "Immunochemical methods in cell and molecular biology" Academic Press, London (1987); or in the series "Methods in Enzymology", Academic Press, Inc. Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.
The gist of this invention is based on the provision of a distinct composition of molecular markers derived from OPA1, i.e. OPA1 isoforms as defined herein, whereby these molecular markers (namely OPA1-L1, -L2, -S3, -S4, -S5) can be measured and analyzed by means and methods provided herein and wherein the composition of the totality of these molecular markers differ between samples obtained from healthy individuals and samples obtained from individuals suffering from or prone to be suffering from a mitochondrial dysfunction or a disorder correlated with mitochondrial dysfunction(s). The composition of molecular markers provided in context of this invention comprises, inter alia, two large isoforms of OPA1 (OPA1-L1, -L2) and three small isoforms of OPA1 (OPA1-S3, -S4, -S5), whereby in particular the ratio between large and small isoforms differs in samples derived from healthy individuals or controls in comparison to samples derived form patients suffering form the mitochondrial dysfunction/disease or patients susceptible to such a disease ("affected patients"). The corresponding novel and inventive finding relates to the fact that in samples derived from individuals who are susceptible for, have predisposition for or have a disorder correlated with mitochondrial dysfunction or mitochondrial disease, the amount of "large isoforms" (OPA1-L1/-L2) is reduced whereas the amount of "small isoforms" (OPA1- S3,-S4, -S5, in particular OPA1-S3 and -S5 and more particular OPA1-S5 as defined herein) is increased. As documented, herein one measure for the determination the susceptibility for, predisposition for or the presence of a disorder correlated with mitochondrial dysfunction or mitochondrial disease is the amount or ratio of the small isoforms versus the large isoforms. Accordingly, the composition of molecular weight markers as provided herein preferably relates to the OPA1 markers in form of OPA1 isoforms (OPA1-L1, -L2, -S3, -S4, -S5 as defined herein), whereby in particular a ratio of more than 10 %, preferably of more than 15%, more preferably of more than 20 % and most preferably of more than 25% of OPA1-S5 in the total amount of all isoforms (OPA1-L1, -L2, -S3, -S4 and -S5) (as evalutated in e.g. SDS-gels or Western-blots) is diagnostic for the susceptibility for, predisposition for or the presence of a disorder correlated with mitochondrial dysfunction or mitochondrial disease.
Accordingly, as a cut-off and-read-out for the evaluation of normal/healthy versus diseased status or susceptibility, the following values can be taken (as non-limiting examples) when for example the five molecular markers as provided herein are analysed in gels (SDS-gels or Western blots derived from said SDS-gels):
For the ratio between OPA1-S5 and all OPA1 isoforms: More than 10%, preferably more than 15%, more preferably more than 20% and more preferably more than 25%. For the ratio between OPA1-S4 and all OPA1 isoforms: More than 25%, preferably more than 27.5% and more preferably more than 30%. For the ratio between OPA1-S3 and all OPA1 isoforms: More than 10%, preferably more than 15% and more preferably more than 20%. For the ratio between OPA1-S3, -S4 and - S5 and all OPA1 isoforms: More than 50%, preferably more than 70% and more preferably more than 90%. For the ratio between OPA1-S3 and -S5 and all OPA1 isoforms: More than 20%, preferably more than 40% and more preferably more than 60%.
Therefore, the present invention also relates, in one embodiment, to a distinct composition of molecular markers, e.g. on a Western blot or in an SDS-PAGE, which comprise the following bands on said SDS-PAGE or Western blot or proteins corresponding to said bands:
An OPA1-L1 band that has an apparent molecular weight of about 97kD (96.8 kD), an OPA1-L2 band that has an apparent molecular weight of about 92 kD (92.3 kD), an OPA1-S3 band that has an apparent molecular weight of about 88 kD (88.1 kD), an OPA1-S4 band that has an apparent molecular weight of about 84 kD (84.4 kD), and/or an OPA1-S5 band that has an apparent molecular weight of about 81 kD (80.9 kD), and wherein the OPA1-S5 band comprises more than 15% +/- 3%, preferably +/- 2%, more preferably +/- 1 % of the density ratio/amount of the all herein defined bands (OPA1-L1, -L2, -S3, -S4 and -S5). In a sample derived from an healthy individual said density ratio/amount is lower than 15% +/- 3%, preferably +/-2%, more preferably +/- 1 %.
In context of this invention, in particular this embodiment, the term "density ratio" or "ratio" means the value of measured amount, e. g. of at least one OPA1 isoform compared to at least one other (or also the same, see above) OPA1 isoform, which can be deduced by standard methods known in the art and which are normally based on optical (also computer-assisted, like image analysis software) measurements. These measurements are also described in the appended examples and comprise scanning of westem-blots and densitometric analysis using standard image software, densitometry and spectrometry.
Therefore, it is within the routine skills of the artisan to detect and deduce the individual OPA1 isoforms protein levels (e.g. the optical density ratio), through densitometry, spectrophotometry and the like.
Exemplarily, the present invention is advantageous in context of high throughput approaches (HTS-methods), such as high throughput assays for determining the ratio of at least one OPA1 isoform as defined herein. Said methods are useful for the determiantion and /or evaluation of mitochondrial dysfunctions and may, therefore, be employed to detect and/or diagnose a mitochondrial dysfunction, a disorder correlated with mitochondrial dysfunction or a mitochondrial disease. This particular advantage is, inter alia, due to the fact that the methods provided herein are fast, robust and easy to perform. The skilled person is readily in a position to apply the experimental approaches performed and discosed herein in the appended examples to corresponding high throughput approaches. "High throughput approaches", which can be employed in context of the present invention are known in the art.
A further aspect disclosed herein relates to a method of screening for a compound capable of (specifically) modulating the activity of proteolytic processing of OPA1. Said method comprises the steps of

(a) contacting OPA1 or a biological system comprising OPA1 with said compound to be screened under conditions allowing proteolytic processing of OPA1 to occur, and
(b) evaluation whether proteolytic processing of OPA1 is altered compared to a control, wherein OPA1 is present and said compound to be screened is absent under conditions allowing proteolytic processing of OPA1 to occur.

In context of this method, the definitions given herein above with respect to terms like "OPA1 processing" or "processing of OPA1" apply here, mutatis mutandis, with respect to the term "proteolytic processing of OPA1". As mentioned above, the process of "OPA1 processing", "processing of OPA1" or "proteolytic processing of OPA1" is characterized by a decrease of the amount of large isoforms of OPA1, an increase of the amount of small isoforms of OPA1 and/or a decrease of the ratio of large versus small isoforms of OPA1 compared to the control. An "altered proteolytic processing of OPA1" as used in context of the above method of screening is, for example, characterized by an altered amount of large and/or small isoforms of OPA1 and/or an altered ratio of large versus small isoforms of OPA1 compared to the control. All corresponding definitions given herein above with respect to the diagnostic methods apply here, mutatis mutandis. Particularly, the large and small isoforms to be employed in context of the method for screening are defined as mentioned herein above. The biological system as employed in context of the herein disclosed screening method can be established by the skilled person. For example, such system may be a cellular system, like those disclosed herein (for example HeLa cells), where OPA1 processing occurs endogenously, or a cellular system, like those disclosed herein (for example yeast cells), where certain components to obtain a condition allowing proteolytic processing of OPA1 to occur are expressed; at least partially, heterologuously.
Based on the teaching provided herein, the skilled person is readily in the position to establish particular methods of screening. For this purpose, the use of a yeast expression system, for example a yeast expression system like the one exemplarily disclosed herein and described in the appended examples, can be of particular benefit. However, the method of screening, is not limited to the use of such an expression system. The skilled person is able to find further expression systems to be employed in context of the method of screening provided herein.
The present invention is further described by reference to the following non-limiting figures and examples.
The Figures show:

Figure 1. Protein sequence alignment of OPA1 spliceforms.

Amino acids encoded by alternative exons 4 (bold), 4b (bold/cursive), and 5b (bold/underlined) are indicated. The amino acid position of each isoform is indicated on the right.

Figure 2. Determination of the apparent molecular weight of OPA1 isoforms by 10% SDS-PAGE.

A, Lane 2, 4, 6: Protein extract from isolated HeLa mitochondria; lane 1: Biorad prestained molecular weight marker; lane 3 PeqLab LMW molecular weight marker; lane 5: Sigma HMW molecular weight marker. Molecular weight of marker proteins ( lane 1, 3, 5) is given in kD. OPA1 isoforms with apparent molecular weight larger than 91 kD are defined as large OPA1 isoforms (I-OPA1#1 and I-OPA1#2). OPA1 isoforms with apparent molecular weight smaller or equal than 91 kD are defined as small OPA1 isoforms (s-OPA1#3, s-OPA1#4 and s-OPA1#5). The dotted white line indicates the 91.0 kD boundary.
B, Molecular weight of all bands representing OPA1 isoforms ( lanes 2, 4 and 6 of A) were calculated from logarithmic regression analysis of migration length versus molecular weight for all shown marker bands ( lanes 1, 3 and 5 of A).

Figure 3. Identification of OPA1 isoforms by mass spectrometry.

a, Critical peptides for assigning the OPA1 spliceform 1 to 8 and the OPA1 isoforms (I-OPA1#1, I-OPA1#2, s-OPA1#3, s-OPA1#4-, s-OPA1#5) according to a first estimation are indicated. Predicted peptides for the eight OPA1 spliceforms shown, were generated by the program Peptidecutter (http://www.expasy.org/tools/peptidecutter; more sophisticated model; Trypsin, lowest cleavage probability 10%). Masses given are M, not M+1 or M-1. In 'times new roman': peptides important to determine the starting point of the mature OPA1 proteins; In 'arial': peptides important to determine the spliceform; in 'courier' : peptides common to all forms of OPA1; In bold: Most N-terminal peptides found in I-OPA1 #1 and in I-OPA1 #2 but not in any s-OPA1 isoform; Underlined: Most N-terminal peptide found in s-OPA1 #3; Bold framed: Most N-terminal peptide found in] s-OPA1 #4; In italic: Most N-terminal peptide found in s-OPA1 #5. Position of cleavage site gives most C-terminal amino acid of respective peptide. Peptides comprising amino acids before position 102 were not detected in this experiment.
b, Immunoprecipitation of OPA1 from isolated HeLa mitochondria (15 mg) using anti-OPA1 antibodies. Equal fractions (0.25%) of total mitochondria (T), supernatant (S) and pellet (P) of solubilized mitochondria after clarifying spin, flow through (FT) and 1% of the elution fraction (E) were analyzed by SDS-PAGE and immunoblotting with anti-OPA1 antibodies.
c, Coomassie stained bands of immunoprecipitated OPA1 isoforms. Each band was separately cut out and used for ESI-LC-MS/MS.
d, Alignment of the N-termini of the eight OPA1 splice variants. The predicted MPP cleavage sites N-terminal to T95 or, alternatively, F88 are indicated. Vertical dotted lines indicate the exon boundaries. Grey highlighted areas represent hydrophobic stretches called TM1, TM2a and TM2b (Herlan 2004, J Cell Biol 165, 167-173). Boxes represent peptides found by ESI-LC-MS/MS analysis of any of the different immunoprecipitated OPA1 isoforms according to the herein disclosed refined MS analysis. The absence (-) or presence (+) of each peptide in different OPA1 isoforms (L1 to S5) is indicated below the alignment. The confidence of peptide identification is indicated by '+++' (excellent), '++' (very good), or '+' (good) based on the number of identifications in separate ESI-LC-MS/MS runs and on the ion score.
e, OPA1 splice variant 7 (Sp.7) was expressed in wild type yeast cells. Total yeast extracts and mitochondria isolated from HeLa cells (M) were analyzed by western blotting with anti-OPA1 antibodies. Bands are labelled according to apparent corresponding size of OPA1 isoforms in HeLa mitochondria (L1, L2, S3, S4, and S5). Precursor protein (P) and a degradation product (d) are indicated.

Figure 4. Model of OPA1-mediated fragmentation of mitochondria in mitochondrial dysfunction and apoptosis.

Mitochondrial dysfunction as observed in a number of human diseases leads to impairment of bioenergetic competence of mitochondria. The reduction of the membrane potential and/or ATP level in mitochondria leads to a proteolytic breakdown of large isoforms of OPA1 to short isoforms. As a consequence, fusion of mitochondria is blocked, and in the presence of ongoing fission, such mitochondria become fragmented. In this way, dysfunctional mitochondria become segregated from the intact network. This may serve two purposes. First, such dysfunctional mitochondria may be removed from cells by autophagy, a process also termed mitoptosis (Skulachev, 2004, Mol Cell Biochem, 256-257, 341-358). Second, a spatial separation as well as a removal would reduce further damage to non-mutated mtDNA caused by ROS produced by a damaged respiratory chain. This mechanism proposes inactivation of OPA1 as a key regulatory step (grey) in counterselecting against damaged mitochondria within cells. The same regulatory step appears to have an early and essential role in apoptosis (dotted arrow lines). Induction of apoptosis, e.g. by staurosporine, activates the proteolytic cleavage of large isoforms of OPA1 to short isoforms. This leads to a block of mitochondrial fusion and fragmentation prior to cytochrome c release. Later in apoptosis cytochrome c and other proapoptotic factors are released from mitochondria and the membrane potential is reduced, leading to further increase of OPA1 processing and mitochondrial fragmentation before cells finally undergo apoptosis.

Figure 5. Mitochondrial fragmentation and alteration of OPA1 protein isoforms precede cytochrome c release in apoptosis.

HeLa cells were treated with 1 µM staurosporine for the indicated time periods, stained with MitoTracker (red) and with cytochrome c antibodies (green).

a, Merged (top row; bottom row) or separately green (2^nd row) and red (3^rd row) confocal fluorescence images. Top, overview (scale bar 20 µm); bottom, indicated box, (scale bar 10 µm).
b, Cells were classified. Tubular, at least one mitochondrial tubule of 5 µm or more; intermediate, at least one between 0.5 and 5 µm but none more than 5 µm; fragmented, none of more than 0.5 µm in length. A representative experiment out of three is shown.
c, Cells were classified for cytochrome c release by immunostaining. Not released, completely colocalised with MitoTracker; intermediate, partly co-localised with MitoTracker; and released, not co-localised with MitoTracker.
d, OPA1 protein isoforms were determined by western blot analysis of total cell extracts. Total cell extracts (C) and isolated mitochondria (M) from untreated HeLa cells were used as controls.

Figure 6. MERRF cybrid cells show fragmentation of mitochondria and alterations in OPA1 isoforms.

MERRF and control cybrid cell lines were cultured, stained either with MitoTracker (red) and immunostained against cytochrome c (green) or with MitoTracker (red) and DAPI (blue).

a, Merged confocal fluorescence images are shown (1^st column: red and green; 2^nd column: red and green; 3^rd column: red and blue; 4^th column: red and blue. Top, overview (scale bar 20 µm); bottom, indicated box, (scale bar 10 µm).
b, Quantification of mitochondrial morphology in cells: Tubular, at least one mitochondrial tubule of 5 µm or more; intermediate, at least one between 0.5 and 5 µm but none more than 5 µm; fragmented, none of more than 0.5 µm in length. Error bars represent standard deviation of five slides evaluated.
c, OPA1 isoforms by western blot analysis of total cell extracts. In c, HeLa cell extracts are shown for comparison.

Figure 7. Mutator mouse embryonic fibroblasts show fragmentation of mitochondria and alterations in OPA1 isoforms.

Immortalized mouse embryonic fibroblasts from two control mice and two mutator mice were cultured, stained either with MitoTracker (red) and immunostained against cytochrome c (green) or with MitoTracker (red) and DAPI (blue).

a, Merged confocal fluorescence images are shown (1^st column: red and green; 2^nd column: red and green; 3^rd column: red and blue; 4^th column: red and blue. Top, overview (scale bar 20 µm); bottom, indicated box, (scale bar 10 µm).
b, Quantification of mitochondrial morphology as in Fig.6b. Error bars represent standard deviation of five slides evaluated.
c, OPA1 isoforms by western blot analysis of total cell extracts.

Figure 8. Patterns of OPA1 isoforms are altered in tissue samples exemplary of mitochondrial dysfunction.

a, Western blot analysis of OPA1 isoforms of heart tissue from mice with a heart-specific knock-out of TFAM (TFAM ko) at 4 or 8 weeks age and of control mice.
b, Homogenates from skeletal muscle biopsies from control individuals (A-D, see Tab.2) and from patients suffering from respiratory disorders (1-10, see Tab.2) analyzed by western blotting for OPA1. The smallest form of OPA1, OPA1-S5 ("s-OPA1"), detected is indicated (arrow).
c, Relative amounts of the smallest OPA1 isoform OPA1-S5 of all OPA1 isoforms determined densitometrically from panel b and from HeLa cells treated or not for 6 h with CCCP (see Fig.9a, two left lanes).

Figure 9. Mitochondrial fragmentation and OPA1 processing after dissipation of the membrane potential are causally linked.

a, HeLa cells and human fibroblasts were treated with CCCP (20 µM) for 6 h and total cell extracts were subjected to western blotting with OPA1 antibodies b-d, Cells were treated or not (Control) with CCCP (20 µM) for 30 min, washed, and incubated further with medium lacking CCCP for the indicated time periods or CCCP (20 µM) was left for 6 h. In parallel, these experiments were performed in the presence of cycloheximide (CHX; 175 µg/ml). Cells were stained with MitoTracker (red) and with cytochrome c antibodies (green).
a', HeLa cells were treated or not with CCCP (20 µM) for 30 min and total cell lysates were subjected to western blotting with the indicated antibodies.
b, Merged confocal fluorescence images are shown. Top, overview (scale bar 20 µm); bottom, indicated box, (scale bar 10 µm).
c, Quantification of mitochondrial morphology as in Fig.6b.
d, OPA1 isoforms by western blot analysis. Extracts of untreated cells (C) and of cells treated for 6 h with CHX alone (+CHX 6 h) were used as controls.
e, HeLa cells were transfected with a plasmid expressing mitochondrial GFP (control), or co-transfected with this plasmid and a plasmid expressing either a mouse isoform of OPA1 corresponding to spliceform 1 (OPA1 ↑), or a dominant negative variant of DRP1 (DRP1_K38E ↑). 36 h after transfection, cells were treated with CCCP for 30 min or not, fixed, and immunostained with GFP (green) and cytochrome c antibodies (red). Merged confocal fluorescence images. Left, overview (scale bar 20 µm); right, indicated box (scale bar 10 µm).
f, Mitochondrial morphology of mitochondrial GFP positive cells was quantified. Cells with at least one highly elongated mitochondrion of more than 10 µm (highly tubular) were quantified in addition to those classes described in Fig.6b.
g, Pulse-chase experiment; HeLa cells were transfected as described herein. 24 h after transfection, cells were subjected to radioactive labeling and subsequent CCCP treatment during the chase period as described for Fig. 11. At indicated times after addition of CCCP, cells were washed, harvested, lysed and subjected to immunoprecipitation with antibodies raised against OPA1 in the presence of 5 mM o-phenathroline / 10 mM EDTA. OPA1 isoformsin the elution fractions were separated by SDS-PAGE and analyzed by digital autoradiography.

Figure 10. Processing and membrane association of OPA1 isoforms.

a, The protease inhibitor o-phenanthroline (o-Phe) and partially DCI (3,4-Dichloroisocoumarin), but not phenylmethylsulphonylfluoride (PMSF) block uncoupler induced conversion of larger OPA1 isoforms to smaller isoforms. HeLa cells were preincubated for 10 min with or without the protease inhibitors at the indicated concentration before CCCP (20 µM) was added. Cells were further incubated for 25 min before they were harvested, lysed in loading buffer, loaded on a SDS-PAGE, and immunoblotted with OPA1 antibodies.
b, The larger isoforms of OPA1 behave in salt and carbonate extraction experiments as integral inner membrane proteins, whereas the smaller isoforms behave like peripherally attached proteins. Shorter isoforms remain peripherally attached to mitochondria even after cells were pretreated with CCCP for 30 minutes (right part). Isolated mitochondria from HeLa cells treated with CCCP for 30 minutes (right) or untreated (left) were extracted either with 30 mM or 500 mM KCl after sonication or with 0.1 M sodium carbonate (pH 11). Pellets were recovered by centrifugation (130,000g, 30 min, 4°C). 100 µg of mitochondrial proteins were used for extraction and 25 % of each pellet (P) and supernatant (S) was analyzed by SDS-PAGE and immunoblotting for the indicated proteins.
c, OPA1 isoforms remain in the mitochondrial fraction independent of pretreatment of cells with CCCP. In addition, neither degradation nor release of other mitochondrial proteins (Tim44, Tim23, AIF, cytochrome c) was observed. As cytosolic marker protein β-actin was used. Hela cells were treated with CCCP as described in b, and subjected to subcellular fractionation. Equal proportions of the mitochondrial fraction (M) and of the cytosolic fraction (C) are analyzed by SDS-PAGE and immunoblotted with indicated antibodies. In total less material was loaded from the CCCP treated derived fractions.

Figure 11. Pulse-chase experiment.

HeLa cells were subjected to radioactive labeling of newly synthesized proteins. After labeling cells were washed, incubated for the indicated time in the absence of radiolabeled amino acids (chase) either in the presence or the absence of CCCP (20 µM). Cells were lysed at indicated times of chase and subjected to immunoprecipitation with antibodies raised against OPA1. Elution fractions were analyzed by digital autoradiography (top panel) and the same membrane was subjected to western blot analysis using OPA1 antibodies (bottom panel). The different OPA1 isoforms are indicated by arrows and named L1-, L2-, S3-, S4- and S5-OPA1.
The Examples illustrate the invention.

Example 1: Material and Methods

Cell culture and reagents:

If not described otherwise, HeLa cells, human fibroblasts, immortalised mouse embryonic fibroblast from control (MEF 13, 14) and mutator mice (MEF 2, 7) (Trifunovic, 2004, Nature 429, 417; Trifunovic, 2005, Proc Natl ), and cybrid cell lines (pT1, pT3) (Chomyn, 1991, Mol Cell Biol 11, 2236) were grown under standard conditions in Dulbecco modified Eagle's medium (DMEM) containing 4.5 g/l glucose and 2 mM L-glutamine supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 50 µg/ml streptomycin. Cell culture reagents were obtained from PAA laboratories (Cölbe, Germany), CCCP, o-phenanthroline and cycloheximide were purchased from Sigma (Germany), DCl from Sigma (Germany), and PMSF from Serva (Germany). The OPA1 plasmid (pMSCV-OPA1) was a kind gift of Luca Scorrano (Padova, Italy) (Cipolat, 2004, Proc Natl Acad Sci USA). The DRP1_K38E N-terminally fused to CFP (pECFP-C1-DVLP_K38E) and mitochondrially targeted GFP (pcDNA3-pOCT:GFP) plasmids were kind gifts of Heidi McBride (Ottawa Heart institute, Canada; Neuspiel, 2005, J Biol Chem, 280, 25060-70; Harder, 2004, Curr Biol, 14, 340-5). Transient transfections of HeLa cells were performed using Metafectene (Biontex Laboratories, Germany). Mitochondria were prepared from HeLa cells by differential centrifugation as described herein.

Yeast plasmids, strains and growth conditions:

OPA1 splice variant 7 was amplified from human cDNA and cloned into pYES2 (Invitrogen, USA). All sequences were verified by DNA sequencing. Haploid W303 was used as wild type control elsewhere. Cells were grown under standard conditions (Guthrie, ). The strain expressing OPA1 was cultured using 2% galactose and 0.5% glucose.

Tissue samples:

Heart tissue was obtained from heart-specific TFAM knockout mice as described earlier (Hansson, 2004, Proc Natl ). Skeletal muscle biopsies were derived from patients diagnosed with respiratory chain disorders or control patients with no such defects (see Tab.2). Informed consent was given by all patients.

Antibodies for immunoblotting:

Anti-OPA1 antibody was affinity purified from a rabbit polyclonal serum raised against the C-terminus of human OPA1 using synthetic peptide: CDLKKVREIQEKLDAFIEALHQEK (SEQ ID No: 29) (Pineda Antikörper-Service, Berlin; BD Biosciences). Antibodies against human MIA40 were raised in rabbits using purified MIA40 fused to MBP. Polyclonal rabbit sera against hTim44, and hTim23 were raised in rabbits as described Bauer (1999, J Mol Biol 289(1), 69-82). Antibodies against AIF (goat anti-AIF, D-20:sc-9416, Santa Cruz Biotechnology, USA), rabbit anti-Fis1 sera (IMGENEX), cytochrome c (mouse, clone 7H8.2C12, BD Biosciences), anti-DRP1 (DLP1 clone 8; BD Biosciences) and β-actin (clone AC-15, Sigma, Germany) were used according to the manufacturer's instructions. The anti-Mfn2 serum was a kind gift of Antonio Zorzano (University of Barcelona, Spain; Pich Hum Mol Genet 2005 14(11):1405-1415).

Fluorescence microscopy:

Live cells were fluorescently labeled with MitoTracker® Red CMXRos (Molecular probes, USA) for mitochondria or with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular probes, USA) for the nucleus, subsequently fixed, and permeabilised. Immunostaining was carried out with mouse anti-cytochrome c monoclonal antibody (clone 6H2.B4; BD Biosciences) and chicken anti-GFP antibody (Aves Lab Inc., USA). The following secondary antibodies conjugated to fluorescent dyes were used: Alexa Fluor^® 488 anti-mouse IgG (H+L) (Molecular probes, USA), Cy3-conjugated anti-mouse IgG and Fluorescein(FITC)-conjugated anti-chicken IgG (Jackson Immunoresearch, USA). Cells were mounted with Prolong® Gold antifade reagent (Molecular probes, USA). All treatments were done according to the manufacturers' instructions. Mitochondrial morphology was analysed by confocal microscopy using a Zeiss LSM 510 (Carl Zeiss Microscopy, Jena, Germany) equipped with a 63x objective. For all imaging, 512x512 pixel images of single confocal planes were acquired and processed with the Bitplane Imaris 4 software.

Example 2: Detection of OPA1 isoforms by SDS-PAGE

Cell pellets were lysed in Lämmli-buffer 1x and heated for 5 min at 95°C. Samples equivalent to ~10⁶ cells were loaded on a 10% acrylamid gel (12 cm high x 16 cm wide plates separated by 1.5 mm thick spacers). Gels were run for 3.5 hours at constant 30 mA or until the 37 kD band of the BioRad Precision Plus Protein standard reaches the bottom of the gel. The proteins were transferred to a nitrocellulose membrane by semi-dry blotting for 1.5 hours at 200 mA using Tris/glycine buffer. The membrane was incubated in TBS buffer containing 5% non fat milk powder for 30 min. The membrane was then incubated in TBS buffer containing 5% non fat milk powder and affinity purified rabbit OPA1 antibodies (raised against the C-terminal peptide of OPA1: DLKKVREIQEKLDAFIEALHQEK (SEQ ID No: 30)) diluted 1:500 for 1 hour. After three rapid washes to remove the primary antibody in excess, the membrane was further incubated for 1 hour in TBS containing 5% non fat milk powder and secondary antibodies anti rabbit conjugated to HRP (horseradish peroxidase) diluted 1:10,000 (BioRad). The membrane was then washed in TBS, once rapidly, and four more times for 5 min each. ECL reagent (enhanced chemoluminescence reagent) was used to reveal the different OPA1 isoform bands on the membrane. Expositions of 1 to 2 min were usually sufficient to detect all OPA1 forms. The compositions of the buffers and gels to be employed are as follows.

4X Lämmli buffer 100ml:

8g SDS
40ml Glycerin
40 min 0.6M Tris pH 6.8
80mg Bromophenol blue
Adjust the volume to 80ml with distilled water and then add
β-Mercaptoethanol 20ml

SDS-PAGE running buffer (electrophoresis buffer) 10X for 5I:

50g SDS
150g Tris
720g Glycine
Add distilled water to reach 5 I

10% Separating gel (10%):

Acrylamide 30% / N, N'-Methylene-bis-acrylamide 0.5 %	5.7 ml
1M Tris pH 8.8	6.5 ml
Distilled water	4.5 ml
TEMED
	10 µl
10%APS	100 µl
Total (for one gel)	17 ml

Stacking gel:

Acrylamide 30% / N, N'-Methylene-bis-acrylamide 0.5 % 17 ml

1 M Tris-HCl pH 8.8 6 ml

10% SDS 1 ml

Distilled water

76 ml

Total (for 20gels) 100 ml

Store at 4°C. Take 5ml for one gel.
Add 25µl 10%APS
5µl TEMED

1X blotting buffer:

20mM Tris	4.84g
150mM Glycine	22.52g
20% Methanol	400ml
0.08% SDS	1.6g
Total	2l (adjusted with distilled water)

10X TBS:

100mM Tris 60.57g

9% NaCl 450g

Total 5l (adjusted with distilled water)

Adjusted to pH 7.4 with HCI.

Example 3: Identification of OPA1 peptides by mass spectrometry

Cells were lysed in lysis buffer (0.5% TritonX100, 150 mM NaCl, 10 mM Tris/HCl pH 7.5, 5 mM EDTA, supplemented with complete protease inhibitor cocktail; Roche, Switzerland) and subjected to standard immunoprecipitation. Thereby, OPA1 isoforms from isolated HeLa mitochondria were immunoprecipitated using an antibody raised against the C-terminal peptide of OPA1 covalently coupled to Sulfo-Link sepharose beads (Pierce, USA) or, alternatively, coupled to Protein A Sepharose CL-4B beads (Amersham Biosciences). If not described otherwise, elution was performed with 1x Lämmli-buffer, separated by SDS-PAGE, stained with Coomassie Blue and bands were cut after in-gel digest with trypsin. All bands were clearly identified as derived from OPA1. Cut gel slices from SDS-PAGE were washed 2x with water and 2x with 40 mM ammonium bicarbonate for 30 min each. After 2x 5 min treatment with 50% acetonitrile, trypsin (Sequencing Grade Modified, Promega) was added and proteins were digested over-night in 40 mM ammonium bicarbonate at 37°C while shaking (650 rpm). Peptides were directly analyzed by MALDI-MS or LC-MS/MS. For example, if not described otherwise, peptides were directly analyzed by nano-ESI-LC-MS/MS for which they were separated on a C18 reversed phase column (75 µm i.d. x 15 cm, packed with C18 PepMap™, 3 µm, 100 A by LC Packings) via a linear acetonitrile gradient, MS and MS/MS spectra were recorded on a QSTAR XL mass spectrometer (Applied Biosystems), and analyzed via the Mascot™ Software (Matrix Science) using the NCBl^nr Protein Database.
The theoretical molecular weight for a corresponding amino acid sequence was calculated using the program "pi_tool" (http://www.expasy.ch/tools/pi tool.html; Bjellqvist, Electrophoresis 1993, 14, 1023-1031; Bjellqvist, ; Gasteiger, 2005, Protein Identification and Analysis Tools on the ExPASy Server, (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press). Thereby, the option "average" regarding the resolution was used.
The corresponding results from a first estimation are as follows:
In the protein bands corresponding to I-OPA1#1 and #2, as most N-terminal peptides, the following peptides were found, besides various peptides present in all OPA1 spliceforms:

YLILGSAVGGGYTAK (SEQ ID NO: 17)
TFDQWK (SEQ ID NO: 18)
DMIPDLSEYK (SEQ ID NO: 19)
WIVPDIVWEIDEYIDFEK (SEQ ID NO: 20)
LAPDFDK (SEQ ID NO: 21)
IVESLSLLK (SEQ ID NO: 22)
ALPNSEDLVK (SEQ ID NO: 23)
DFFTSGSPEETAFR (SEQ ID NO: 24)

Figure 1

3a

Accordingly, in large OPA1 isoforms at least the amino acids from position 102 to the most C-terminal amino acid of all OPA1 spliceforms (SEQ ID No:2, 4, 6, 8, 10, 12, 14, 16) are comprised and the theoretical molecular weights vary between 89 und 99 kD, depending of the corresponding spliceform to be used for molecular weight calculation.
Based on in silico predictions (prediction of cleavage of the leader sequence by mitochondrial processing peptidase (MPP) using the computer program Mitoprot (Claros, 1996, Eur J Biochem, 241, 779-86)), it was deduced that large OPA1 isoforms already start with amino acid position 95 of all OPA1 spliceforms (SEQ ID No:2, 4, 6, 8, 10, 12, 14, 16), and hence, start with the peptide TRLLKLRYLILGS (SEQ ID NO: 25) .... According to this finding, the above mentioned theoretical molecular weights are increased by 0.9 kD.
None of the above mentioned peptides were detected in protein bands corresponding to small isoforms of OPA1.
Further, in I-OPA1#1 but not in I-OPA1#2 the following peptides were found: GLLGELILLQQQIQEHEEEAR (SEQ ID NO: 26) and AAGQYSTSYAQQK (SEQ ID NO: 27).
From the data provided in the first estimation, it can be deduced that I-OPA1#1 be derived from spliceform 7 and I-OPA1 #2 be derived from spliceform 1.
In the protein band corresponding to s-OPA1#3, as most N-terminal peptide, the peptide GLLGELILLQQQIQEHEEEAR (SEQ ID NO: 26) was found. Moreover and inter alia, the peptides AAGQYSTSYAQQK (SEQ ID NO: 27) and IDQLQEELLHTQLK (SEQ ID NO: 28) were found. It was deduced that s-OPA1#3 could be derived from spliceforms 4, 6, 7, and/or 8 and that the corresponding most N-terminal amino acid is, dependent on the amino acid sequence of the mentioned spliceforms, at position 173 (SEQ ID No: 10), 191 (SEQ ID No: 6), 209 (SEQ ID No: 4) and 227 (SEQ ID No: 2), respectively. The theoretically predicted molecular weight of the s-OPA1#3 is about 91.8 kD.
In the protein band corresponding to s-OPA1#4, as most N-terminal peptide, the peptide AAGQYSTSYAQQK (SEQ ID NO: 27) was found. Moreover and inter alia, the peptide IDQLQEELLHTQLK (SEQ ID NO: 28) was found. It was deduced that s-OPA1#4 could derive from spliceforms 4, 6, 7, and/or 8 and that the corresponding most N-terminal amino acid is, dependent on the amino acid sequence of the mentioned spliceforms, at position 195 (SEQ ID No: 10), 213 (SEQ ID No: 6), 231 (SEQ ID No: 4) and 249 (SEQ ID No: 2), respectively. The theoretically predicted molecular weight of the s-OPA1#4 is about 89.2 kD.
In the protein band corresponding to s-OPA1#5; as most N-terminal peptide, the peptide IDOLOEELLHTQLK (SEQ ID NO: 28) was found. It was deduced that s-OPA1#5 could derive from one or more of spliceforms 1 to 8 and that the corresponding most N-terminal amino acid is, dependent on the amino acid sequence of the mentioned spliceforms, at position 215 (SEQ ID No: 16), 179 (SEQ ID No: 14), 197 (SEQ ID No: 12), 216 (SEQ ID No: 10), 233 (SEQ ID No: 8), 234 (SEQ ID No: 6), 252 (SEQ ID No: 4) and 270 (SEQ ID No: 2), respectively.
The theoretically predicted molecular weight of the s-OPA1#5 is about 86.8 kD.
Moreover, in none of the small OPA1 isoforms, the peptide DFFTSGSPEETAFR (SEQ ID NO: 24) was found. Accordingly, is can be deduced that the N-terminal amino acid of each s-OPA1 lies within or shortly C-terminal to this sequence. In either case, the N-terminal amino acid of each small OPA1 lies N-terminal to the above described found most N-terminal peptides of each small OPA1 (see above).

Based on the results from the first estimation with respect to the OPA1 peptides determined by the mass spectrometry experiments, for each OPA1 isoform the following theoretically calculated molecular weights and the apparent starting position in the corresponding spliceform of OPA1 (see also the corresponding SEQ ID No) were deduced:

Isoform (amino acid position start / MW in kD)
Spliceform	I-OPA1#1/#2	s-OPA1#3	s-OPA1#4	s-OPA1#5
8	95 / 105.9 (106,8*)	227 / 91.8	249 / 89.2	270 / 86.8
7	95 / 104.9	209 / 91.8	231 / 89.2	252 / 86.8
6	95 / 102.7	191 / 91.8	213 / 89.2	234 / 86.8
5	95 / 102.5	x	x	233 / 86.8
4	95/100.8	173/91.8	195/89.2	216 / 86.8
3	95 / 98.5	x	x	197 / 86.8
2	95/96.6	x	x	179 / 86.8
1	95 / 100.7	x	x	215 / 86.8

To obtain an averaged apparent molecular weight value for either I-OPA1#1 or I-OPA1#2, the four smallest and the four largest molecular weight values of the second column of the above table ("I-OPA1#1/#2") where averaged. Accordingly, for I-OPA1#1 an averaged molecular weight value of 104.0 kD (104,2*) and for I-OPA1#2 an averaged molecular weight value of 99.2 kD was derived. The values indicated by an asterix (*) are based on a repeated estimation.
Alternatively, based the herein provided evindence that I-OPA1#1 is derived from spliceform 7 and that I-OPA1#2 is derived from spliceform 1, for I-OPA1#1 a theoretic molecular weight value of 104.9 kD and for I-OPA1#2 a theoretic molecular weight value of 100.7 kD was calculated.
In the following, the corresponding results from a second, refined analysis are given:
In the protein bands corresponding to I-OPA1#1 and #2, as most N-terminal peptides, the following peptides were found, besides various peptides present in all OPA1 spliceforms:

Figure 1

3d

Accordingly, in large OPA1 isoforms at least the amino acids from position 102 to the most C-terminal amino acid of all OPA1 spliceforms (SEQ ID No:2, 4, 6, 8, 10, 12, 14, 16) are comprised and the theoretical molecular weights vary between 95.7 und 105.9 kD, depending of the corresponding spliceform to be used for molecular weight calculation (see below).
Based on in silico predictions (prediction of cleavage of the leader sequence by mitochondrial processing peptidase (MPP) using the computer program Mitoprot (Claros, 1996, Eur J Biochem, 241, 779-86)), it was deduced in a first estimation that large OPA1 isoforms already start with amino acid position 95 of all OPA1 spliceforms (SEQ ID No:2, 4, 6, 8, 10, 12, 14, 16), and hence, start with the peptide TRLLKLRYLILGS (SEQ ID NO: 25). According to this finding, the above mentioned theoretical molecular weights are increased by 0.9 kD. However, based on an alternative MPP cleavage site present from amino acid 86 to 88 (Taylor Structure. 2001 ), it was deduced in a refined analysis that large OPA1 isoforms already start with amino acid position 88 of all OPA1 spliceforms (SEQ ID No:2, 4, 6, 8, 10, 12, 14, 16), and hence, start with the peptide FWPARLATRLLKLRYLILGS (SEQ ID NO: 111). According to this finding, the above mentioned theoretical molecular weights are increased by 1.7 kD instead of 0.9 kD. None of the above mentioned peptides were detected in protein bands corresponding to small isoforms of OPA1.
Further, in I-OPA1#1 but not in I-OPA1#2 the following peptide was found:

AAGQYSTSYAQQK (SEQ ID NO: 27).

From the data provided in the refined analyses, it can be deduced that I-OPA1#1 be derived from spliceform 7 and I-OPA1 #2 represents a mixture of two isoforms derived from splice variants 1 and 4.
In the protein band corresponding to s-OPA1#3, as most N-terminal peptides, the peptides IVESLSLLK (SEQ ID No: 22), DFFTSGSPEETAFR (SEQ ID No: 24), and GLLGELILLQQQIQEHEEEAR (SEQ ID NO: 26) were found. Moreover and inter alia, the peptides AAGQYSTSYAQQK (SEQ ID NO: 27) and IDQLQEELLHTQLK (SEQ ID NO: 28) were found. It was deduced that s-OPA1#3 is derived from spliceform 7 and that the corresponding most N-terminal amino acid is 172 (SEQ ID No: 4), or close-by more N-terminal to this position. The theoretically predicted molecular weight of the s-OPA1#3 is about 95.9 kD.
In the protein band corresponding to s-OPA1#4, as most N-terminal peptides, the peptides .GLLGELILLQQQIQEHEEEAR (SEQ ID NO: 26) and AAGQYSTSYAQQK (SEQ ID NO: 27) were found. Moreover and inter alia, the peptide IDQLQEELLHTQLK (SEQ ID NO: 28) was found. It was deduced that s-OPA1#4 could derive from spliceforms 4, 6, 7, and/or 8 and that the corresponding most N-terminal amino acid is, dependent on the amino acid sequence of the mentioned spliceforms, at position 173 (SEQ ID No: 10), 191 (SEQ ID No: 6), 209 (SEQ ID No: 4) and 227 (SEQ ID No: 2), respectively. The theoretically predicted molecular weight of the s-OPA1#3 is about 91.8 kD.
In the protein band corresponding to s-OPA1#5, as most N-terminal peptide, the peptide IDQLQEELLHTQLK (SEQ ID NO: 28) was found. It was deduced that s-OPA1#5 could derive from one or more of spliceforms 1 to 8 and that the corresponding most N-terminal amino acid is, dependent on the amino acid sequence of the mentioned spliceforms, at position 215 (SEQ ID No: 16), 179 (SEQ ID No: 14), 197 (SEQ ID No: 12), 216 (SEQ ID No: 10), 233 (SEQ ID No: 8), 234 (SEQ ID No: 6), 252 (SEQ ID No: 4) and 270 (SEQ ID No: 2), respectively.
The theoretically predicted molecular weight of the s-OPA1#5 is about 86.8 kD.
Moreover, in the refined analyses, in none of the small OPA1 isoforms, the peptide LAPDFDK (SEQ ID NO: 21) was found. Accordingly, is can be deduced that the N-terminal amino acid of each s-OPA1 lies within or shortly C-terminal to this sequence. In either case, the N-terminal amino acid of each small OPA1 lies N-terminal to the above described found most N-terminal peptides of each small OPA1 (see above).

Based on the results from the refinded analyses with respect to the OPA1 peptides determined by the mass spectrometry experiments, for each OPA1 1 isoform the following theoretically calculated molecular weights and the apparent starting position in the corresponding spliceform of OPA1 (see also the corresponding SEQ ID No) were deduced:

Isoform (amino acid position start / MW in kD)
Spliceform	I-OPA1#1/#2	s-OPA1#3	s-OPA1#4	s-OPA1#5
8	88 / 107.6	x	227 / 91.8	270 / 86.8
7	88 / 105.8	172 / 95.9	209 / 91.8	252 / 86.8
6	88 / 103.5	x	191 / 91.8	234 / 86.8
5	88 / 103.4	x	x	233 / 86.8
4	88/101.7	x	173/91.8	216 / 86.8
3	88/99.3	x	x	197 / 86.8
2	88/97.4	x	x	179 / 86.8
1	88/ 101.5	x	x	215 / 86.8

To obtain an averaged apparent molecular weight value for either I-OPA1#1 or I-OPA1#2, the four smallest and the four largest molecular weight values of the second column of the above table ("I-OPA1#1/#2") where averaged. Accordingly, for I-OPA1#1 an averaged molecular weight value of 105.1 kD and for I-OPA1#2 an averaged molecular weight value of 100.0 kD was derived.
Altematively, based the herein provided evindence that I-OPA1#1 is derived from spliceform 7 and that I-OPA1#2 is derived from spliceform 1 or spliceform 4, for I-OPA1#1 a theoretic molecular weight value of 105.8 kD and for I-OPA1#2 a theoretic molecular weight value of 101.5 kD or 101.7 kD, respectively, was calculated.
To examine to which extent the large and/or small OPA1 isoforms are generated by alternative splicing or limited proteolysis, mitochondria from HeLa cells were isolated and the different OPA1 species were purified by immunoprecipitation (Fig. 3bc). The antibodies used were directed against a C-terminal peptide of OPA1 present in all OPA1 isoforms. The various species were resolved by SDS-PAGE and analyzed by LC-MS/MS spectrometry (Figure 3d). The most N-terminal tryptic peptide found in L1 and L2 was located a few amino acid residues C-terminal to the cleavage site of the mitochondrial processing peptidase (MPP) between A94 and T95 or, alternatively, N87 and F88 (see above). Specifically for L1 and L2 a number of peptides were found located C-terminally to the MPP cleavage site (Figure 3d). These peptides were derived from exon 3 and from alternatively spliced exons 4 and 5b but not from exon 4b. Small OPA1 isoforms (S3 to S5) were lacking increasingly more of these peptides from the N-terminus (Figure 3d). All S-forms lacked the transmembrane segment TM1. Besides that, numerous peptides corresponding to exon 6-29, which are common to all splice variants, were identified in all isoforms (Figure 3d and data not shown).
In order to assign the isoforms to individual splice variants the presence or absence of peptides in the purified isoforms that cover or overlap exons affected by alternative splicing were taken into account. For L1 peptides that all could be derived from splice variant 7 but not from a different splice variant alone were obtained (Figure 3). The calculated molecular weights of each splice variant when cleaved by MPP are approximately as follows: 101.5 kDa (Sp.1), 97.4 kDa (Sp.2), 99.3 kDa (Sp.3), 101.7 kDa (Sp.4), 103.4 kDa (Sp.5), 103.5 kDa (Sp.6), 105.8 kDa (Sp.7), and 107.6 kDa (Sp.8). Thus, as L2 contained the same nine N-terminal peptides as L1 but was smaller in size (Fig. 3bc) it is unlikely to be derived from splice variant 7 or 8. Further, the peptide pattern found for L2 was consistent with splice variant 1 except for one peptide (GLLGELILLQQQIQEHEEEAR (SEQ ID NO: 26)) which could theoretically be derived from splice variants 4, 6, 7 or 8 (Figure 3d). However, only the predicted size of MPP cleaved splice variant 4 is nearly identical to the one of splice variant 1. Therefore, in HeLa cells L2 most likely represents a mixture of two isoforms derived from splice variants 1 and 4 whereas L1 is derived from splice variant 7 (Figure 3d). These results are consistent with the high level of expression of these variants in HeLa cells (Satoh, Biochem ). S3 contains peptides representing splice variant 7 while S4 contains peptides derived from splice variants 4, 6, 7, or 8, and S5 contains only peptides common to all eight splice variants. In conclusion, these results suggest that S-forms are generated by proteolysis of larger OPA1 isoforms possibly derived from different splice variants.

Example 4: The role of OPA1 during apoptosis

In a first approach to study the molecular mechanism of fragmentation of mitochondria in higher organisms, the role of fusion-promoting OPA1 during apoptosis was investigated. The levels of OPA1 isoforms in HeLa cells after induction of apoptosis were determined. Treatment of cells with staurosporine resulted in rapid fragmentation of mitochondria within 2 - 3 h (Fig. 5ab). This coincided with the disappearance of the two largest OPA1 isoforms and a concomitant increase of small isoforms of OPA1 (Fig.5d). Release of cytochrome c occurred at markedly later time; only after 5 - 6 h had more than 50% of cells released cytochrome c (Fig.5c). Thus, fragmentation during apoptosis occurs earlier than release of cytochrome c and concomitantly with disappearance of larger OPA1 isoforms.

Example 5: Determination of OPA1 isoforms in MERRF cells

In a further approach to study the molecular mechanism of fragmentation of mitochondria in human diseases, the role of fusion-promoting OPA1 in a cybrid cell line derived from a MERRF patient suffering from severe myopathy (Chomyn, 1991, Mol Cell Biol 11, 2236) was investigated. The mtDNA in the cell line contained the A8344G mutation in the tRNA^Lys in a nearly homoplasmic (∼98%) manner leading to a substantial impairment of mitochondrial function (Chomyn, 1991, Mol Cell Biol 11, 2236). The cells from the MERRF patient, but not control cells, showed highly fragmented mitochondria (Fig. 6ab). The pattern of the five detected OPA1 protein isoforms was altered in the MERRF cells compared to control cells (Fig.6c). It appears that in the MERRF cells the larger isoforms are reduced compared to, at least, the smallest isoform of OPA1 (OPA1-S5). As OPA1 is required for mitochondrial fusion the observed loss of large OPA1 isoforms may explain the fragmentation of mitochondria in this model system of mitochondrial dysfunction.

Example 6: Determination of OPA1 isoforms in fibroblasts of the 'mutator mouse'

To further substantiate the findings of Example 5, immortalized mouse embryonic fibroblasts derived from the so-called 'mutator mouse' (Trifunovic, 2004, Nature 429, 417; Trifunovic, 2005, Proc Natl ) were analyzed. This mouse was generated by a homozygous knock-in of a variant of mtDNA polymerase γ resulting in a phenotype of premature aging. The variant enzyme has much reduced proofreading activity and mtDNA accumulates random point mutations at a 3-5 fold higher rate than normal leading to severe mitochondrial dysfunction. Mitochondria were extensively fragmented in mutator but not in control cell lines (Fig. 7ab). The levels of the larger OPA1 isoforms were strongly reduced whereas the levels of the smaller forms were increased in the mutant cells (Fig.7c). These findings suggest that mutations in mtDNA are causative to changes in OPA1 isoform levels and mitochondrial fragmentation.

Example 7: Determination of OPA1 isoforms in heart tissue of TFAM knockout mice

TFAM is an essential mitochondrial transcription factor also required for mtDNA maintenance. A heart-specific knockout of TFAM in mice led to a severe depletion of mtDNA in the heart, resulting in cardiomyopathy and altered mitochondrial morphology (Hansson, 2004, Proc Natl ). The pattern of OPA1 isoforms in heart tissue of these mice was changed as compared to controls; the abundance of large OPA1 isoforms was reduced whereas that of small isoforms was increased (Fig.8a). This was even more pronounced at eight weeks compared to four weeks of age (Fig.8a), consistent with the progression of the cardiomyopathy in those mice (Hansson, 2004, Proc Natl ). This shows that mitochondrial dysfunction resulting from mtDNA depletion is linked to the reduction of large OPA1 isoforms in the affected tissue. Moreover, the alterations of mitochondrial morphology observed earlier by electron microscopy (Hansson, 2004, Proc Natl ) are likely due to the shift in the levels of OPA1 isoforms. In addition, it was observed whether alterations in the OPA1 isoform pattern are detectable in patients diagnosed with respiratory chain defects (Tab.2). Patients 1 to 10 included in this study were previously diagnosed with respiratory chain disorders. All patients suffered from mitochondrial encephalomyopathies or isolated myopathies on the basis of clinical, biochemical, morphological and, in some cases, genetic findings. Skeletal muscle biopsies from patients A to D representing non-mitochondrial disorders served as controls. The activities of the respiratory chain complexes of all control patients were within the normal range. Measurements of rotenone sensitive NADH-ubiquinone oxidoreductase (complex I), succinate-cytochrome c oxidoreductase (complexes II and III) and cytochrome c oxidase (complex IV) were determined spectrophotometrically in skeletal muscle homogenates according to Fischer, 1986, Eur J Pediatr, 144, 441-444, after informed consent was given by the patient. Activities were expressed as units per gram of non-collagenous protein and related to the mitochondrial marker enzyme citrate synthase. A mitochondrial DNA depletion syndrome was diagnosed in patients 1, 4 and 10. In patient 8, a homozygous mutation (G1541A) was identified in the SCO2 gene and in patient 7, mtDNA analysis led to the identification of a heteroplasmic A3243G mutation in the mitochondrial tRNA-Leu_(UUR) gene, the MELAS mutation. Homogenates from skeletal muscle biopsies were analysed by western blotting for OPA1 pattern (Fig.8b). The relative amount of the smallest detected form of OPA1 ("s-Opa1", which is isoform s-OPA1#5) of all OPA1 isoforms was analyzed densitometrically from the immunoblot (Fig. 8bc).

Indeed, an increase of small OPA1 isoforms was observed in skeletal muscle from these patients but not from controls (Fig.8b). The ratios of the levels of the smallest OPA1 isoform ("s-Opa1", which is isoform s-OPA1#5) to the total of all OPA1 isoforms exhibited a much broader variation in patients compared to controls. The patients with mitochondrial DNA depletion syndromes or harboring a MELAS mutation were among those with the highest ratios (Fig.8c, Table below; patients 1 and 7). In none of the controls, such a shift was observed. This demonstrates that OPA1 isoforms are altered in patients with mitochondrial disorders, in particular those with depletion or mutation of the mtDNA.

Samples		Respiratory chain enzyme activities¹		Genetics	% s-OPA1 of total²
A		normal		-	6.7
C		normal		-	7.5
B		normal		-	10.7
D		normal		-	14.7
5	I: 10%			unknown	14.7
9	I: 80%	II/III: 40 %;	IV: 20%	unknown	16.3
2			IV: 20 %	unknown	17.3
8	1: 70%	II/III: 60%	IV: 10%	A1541G mutation in SCO2	18.7
6	1: 30%			unknown	22.1
4	I: 30%		IV: 30%	mtDNA depletion	22.7
3	I: 30%	II/III: 80%	IV: 30%	unknown	22.9
10	I: 50%	II/III: 50%	IV: 20%	mtDNA depletion	23.5
7	I: 50 % I: 50 %	II/III: 50%	IV: 80% IV: 80%	MELAS A3243G mutation in tRNA-Leu^(UUR)	39.7
1	I: 50%	II/III: 50%	IV: 20%	mtDNA depletion	51.2

¹Activities of the respiratory chain complexes I, II/III and IV are expressed in % of the lowest limit of the respective reference range. ²The term "s-OPA1" used in this table denotes the herein definded s-OPA1#5.

Example 8: Determination of ratios of OPA1 isoforms

From the results of the below described uncoupler experiments using HeLa cells (Example 9; Fig.9a), the ratio of the relative amount of various OPA1 isoforms was determined. For this purpose, total protein extracts were separated by SDS-PAGE and immunoblotted using an antibody raised against the C-terminus of OPA1. The western blot was scanned and densitometrically analysed using standard imaging software. The intensity of each band was determined and the background intensity was subtracted. The results so generated are listed in the following table:

Experiment	OPA1#	3+#5 / all bands %	OPA1#	3+#4+#5/ / all bands %	OPA1#	3/ all bands %	OPA1#	4 / all bands %	OPA1#	5 / all bands %
HeLa - CCCP	20.2	47.4	9.9	27.2	10.3
HeLa + CCCP	64.8	93.0	19.2	28.2	45.6
fold increase	3.2	2.0	1.9	1.0	4.4

Example 9: Uncoupler Experiments

It was further observed whether dissipation of the membrane potential is sufficient to trigger changes in abundance of OPA1 isoforms and fragmentation of mitochondria. Indeed, treatment of HeLa cells or fibroblasts with the uncoupler CCCP led to a dramatic shift of OPA1 isoforms towards the smaller isoforms (Fig.8c, 9a). This is apparently due to proteolytic cleavage of the large forms since this process was (at least partially) blocked in the presence of the protease inhibitors o-phenanthroline and DCI (Fig.10a). Further, the small isoforms produced could be extracted from mitochondria with detergent-free buffer with a higher efficiency than the large forms suggesting that the large forms are integral membrane protein isoforms whereas the small ones are only peripherally attached to the membrane (Fig.10b). Proteolytic processing occurs within mitochondria as shown by cellular fractionation experiments (Fig.10c). This is specific for OPA1 since degradation of other mitochondrial proteins is not observed (Fig.10c). Fragmentation of mitochondria occurred rapidly within 15 to 30 min after addition of CCCP (Fig. 9bc). Processing of OPA1 took place within the same narrow time frame (Fig.9d). Impairment of fusion of mitochondria may therefore be due to rapid inactivation of OPA1 by proteolysis of large isoforms. Moreover, upon removal of CCCP normal mitochondrial morphology was recovered and this coincided with the reappearance of large isoforms of OPA1 (Fig. 9cd). Mitochondrial fragmentation and OPA1 processing are not accompanied by cytochrome c release in this or in any of the investigated models of mitochondrial dysfunction (Fig.6a; 7a; 9b; 10c and data not shown). This suggests that mitochondrial fragmentation per se does not result in apoptosis. It was tested whether uncoupler induced fragmentation and reversal of fragmentation require protein synthesis. The protein synthesis inhibitor cycloheximide (CHX) did not interfere with fragmentation and OPA1 processing (Fig. 9cd). This indicates that fragmentation of mitochondria and activation of OPA1 cleavage are independent of protein synthesis. However, recovery of a tubular mitochondrial network as well as the reappearance of large OPA1 isoforms, was impaired in the presence of CHX (Fig. 9cd). Therefore, mitochondrial fragmentation and the shift of OPA1 isoforms from large to small are not reversible without ongoing protein synthesis, consistent with the explanation that a proteolytic inactivation of OPA1 causes mitochondrial fragmentation. In order to show the causal and specific role of OPA1 in this process it was investigated whether OPA1 overexpression can block uncoupler-induced fragmentation. Indeed, fragmentation of mitochondria after CCCP treatment was largely prevented upon overexpression of OPA1 (Fig. 9ef). This suggests that OPA1 is directly involved in the fragmentation of mitochondria induced after loss of the mitochondrial membrane potential. A similar effect was observed after the expression of a dominant-negative variant of DRP1
(DRP1_K38E) that prevents fission of mitochondria (Fig. 9ef). Thus, the fragmentation that occurs by a block of mitochondrial fusion depends on the normal DRP1-dependent fission pathway.
In order to check whether overexpression of OPA1 or of a dominant negative variant of DRP1 (DRP1_K38E) affects the rate of uncoupler-induced conversion of large OPA1 isoforms into smaller OPA1 isoforms a pulse-chase experiment was performed. Indeed, overexpression of OPA1 slowed down processing of OPA1 as indicated by the prolonged presence of L1- and L2-OPA1 when compared to the control overexpression of GFP or of DRP1K38E (Fig. 9g). This effect may even be underestimated since also endogenous OPA1 from about 50 % of nontransfected cells contributes to the signal. Thus, additional expression of large isoforms of OPA1 blocks CCCP-induced mitochondrial fragmentation. A dominant-negative variant of DRP1 (DRP1K38E) does not affect the rate of OPA1 processing but blocks mitochondrial fragmentation (Fig. 9efg). Thus, the fragmentation that occurs by a block of mitochondrial fusion depends on the normal DRP1-dependent fission pathway.

Example 10: Pulse-chase experiments

The large OPA1 isoforms I-OPA1#1 and I-OPA1#2 are converted to the small OPA1 isoforms s-OPA1#3, s-OPA1#4 and/or s-OPA1#5. These findings were demonstrated by a pulse-chase experiment in which HeLa cells were grown under standard growth conditions in 500 µl DMEM w/o methionine/cysteine for 30 min (starvation), then pulse-labelled by the addition of 50 µCi ³⁵[S]-methionine/cysteine (5 µl) for 2.5 hours. Then cells were washed 3 times with PBS and 1 ml fresh DMEM medium + 10 %FCS without radioactive methione (chase) was added. Cells were either treated with 20 µM CCCP for 1 hour or not. OPA1 was immunoprecipitated using an antibody raised against the C-terminus of OPA1 and OPA1 was detected by autoradiography and by western-blotting after SDS-PAGE. By both methods a shift from the large isoforms to the short isoforms was detected upon treatment with CCCP (Fig. 11). Accordingly, it can be concluded that I-OPA1 isoforms are actually converted to s-OPA1 isofoms, in particular towards s-OPA1#5.
Example 11: Determination of the apparent molecular weight of OPA1 isoforms by 10% SDS-PAGE
Mitochondria were prepared from HeLa cells by standard methods using differential centrifugation. For this purpose, cells were, for example, harvested, washed in phosphate-buffered saline supplemented with 5 mM EDTA, and resuspended in 1 ml of RSB buffer (10 mM HEPES (pH 7.5), 1 mM EDTA, 210 mM mannitol, 70 mM sucrose, supplemented with complete protease inhibitor mixture). Mitochondria were prepared after disruption of HeLa cells by passing 6 times through a 26G needle fitted to a 5 ml syringe applying a method adapted from Arnoult, 2005, J Biol Chem, 280(42), 35742-50. Cell pellets from low speed centrifugations (2,000 X g, 10 min, 4 °C) were resuspended in RSB buffer and passaged again through a needle as described. This step was repeated 3 to 4 times. The supernatants from low speed centrifugations were pooled and centrifuged again (13,000 X g, 10 min, 4 °C) to obtain a crude mitochondrial pellet and a cytosolic supernatant. Three aliquots of a protein extract (24 µg each) from isolated HeLa mitochondria were separated by 10% SDS-PAGE as described above (see example 2 and Fig.2). Three different molecular weight markers were loaded next to these (BioRad, Germany, Precision Plus Protein Standard All Blue, #1610373; PeqLab, Germany, Protein MW marker, #27-1010; Sigma, Germany, HMW marker, #M3788). Five OPA1 isoforms with apparent average molecular weights of 96.8 kD, 92.3 kD, 88.1 kD, 84.4 kD, and 80.9 kD were observed (Fig.2). The molecular weight was determined by logarithmic regressions analysis of the migration length all marker bands with known molecular weights, determining the migration length of all OPA1 isoforms, and subsequently calculating the apparent molecular weight in kD. The bands larger than 91 kD are defined as large OPA1 isoforms ((I-)OPA1#1 and (I-)OPA1#2). OPA1 isoforms with apparent molecular weight smaller or equal than 91 kD are defined as small OPA1 isoforms ((s-)OPA1#3, (s-)OPA1#4 and (s-)OPA1#5).
The present invention refers to the following nucleotide and amino acid sequences:

SEQ ID No. 1:
Nucleotide sequence encoding OPA1 spliceform 8 (NM_130837; CDS 56-3103)
SEQ ID No. 2:
Amino acid sequence of OPA1 spliceform 8 (NP_570850; 1015 aa)
SEQ ID No. 3:
Nucleotide sequence encoding OPA1 spliceform 7 (NM_130836; CDS 56-3049)
SEQ ID No. 4:
Amino acid sequence of OPA1 spliceform 7 (NP_570849; 997 aa)
SEQ ID No. 5:
Nucleotide sequence encoding OPA1 spliceform 6 (NM_130835; CDS 56-2995)
SEQ ID No. 6:
Amino acid sequence of OPA1 spliceform 6 (NP_570848; 979 aa)
SEQ ID No. 7:
Nucleotide sequence encoding OPA1 spliceform 5 (NM_130834; CDS 56-2992)
SEQ ID No. 8:
Amino acid sequence of OPA1 spliceform 5 (NP_570847; 978 aa)
SEQ ID No. 9:
Nucleotide sequence encoding OPA1 spliceform 4 (NM_130833; CDS 56-2941)
SEQ ID No. 10:
Amino acid sequence of OPA1 spliceform 4 (NP_570846; 961 aa)
SEQ ID No. 11:
Nucleotide sequence encoding OPA1 spliceform 3 (NM_130832; CDS 56-2884)
SEQ ID No. 12:
Amino acid sequence of OPA1 spliceform 3 (NP_570845; 942 aa)
SEQ ID No. 13:
Nucleotide sequence encoding OPA1 spliceform 2 (NM_130831; CDS 56-2830)
SEQ ID No. 14:
Amino acid sequence of OPA1 spliceform 2 (NP_570844; 924 aa)
SEQ ID No. 15:
Nucleotide sequence encoding OPA1 spliceform 1 (BC075805; CDS 155-3037)
SEQ ID No. 16:
Amino acid sequence of OPA1 spliceform 1 (AAH75805; 960 aa)

SEQUENCE LISTING

<110> Ludwig-Maximilians-Universität München
<120> Method for diagnosing mitochondrial dysfunction
<130> M1590 PCT S3
<160> 111
<170> PatentIn version 3.3
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<400> 95
<210> 96
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(5)
<223> OPA1
<400> 96
<210> 97
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(10)
<223> OPA1
<400> 97
<210> 98
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(8)
<223> OPA1
<400> 98
<210> 99
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(10)
<223> OPA1
<400> 99
<210> 100
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(7)
<223> OPA1
<400> 100
<210> 101
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(17)
<223> OPA1
<400> 101
<210> 102
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(10)
<223> OPA1
<400> 102
<210> 103
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(9)
<223> OPA1
<400> 103
<210> 104
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(12)
<223> OPA1
<400> 104
<210> 105
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(5)
<223> OPA1
<400> 105
<210> 106
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(8)
<223> OPA1
<400> 106
<210> 107
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(5)
<223> OPA1
<400> 107
<210> 108
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(12)
<223> OPA1
<400> 108
<210> 109
<211> 25
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(25)
<223> OPA1
<400> 109
<210> 110
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(11)
<223> OPA1
<400> 110
<210> 111
<211> 20
<212> PRT
<213> artificial sequence
<220>
<223> fragment of OPA1
<220>
<221> source
<222> (1)..(20)
<400> 111

Claims

A method for determining the susceptibility for, predisposition for or the presence of a disorder correlated with mitochondrial dysfunction or mitochondrial disease, comprising the in vitro determination selected from the group consisting of
(a) the in vitro determination of the ratio of at least one large and at least one small isoform of the optic atrophy 1 protein (OPA1), whereby said large isoform has an apparent molecular weight of more than 91 kD and whereby said small isoform has an apparent molecular weight of less than 91 kD, said molecular weights being determined by SDS-PAGE analysis; and/or
whereby said large isoform has an apparent molecular weight of more than 95 kD and whereby said small isoform has an apparent molecular weight of less than 95 kD, said molecular weights being determined by mass spectrometry;

(b) the in vitro determination of (a) wherein the ratio of at least two OPA1 isoforms is determined; and

(c) the in vitro determination of (a) comprising the step of determining the ratio between at least one large OPA1 isoform and an internal standard and between at least one small OPA1 isoform and an internal standard, wherein a decrease of the large OPA1 isoforms accompanied by an increase of the small OPA1 isoforms is diagnostic for said disorder or disease.
The method of claim 1, wherein said large isoforms comprise two isoforms (OPA1-L1 and OPA1-L2) and wherein said small isoforms comprise three isoforms (OPA1-S3, OPA1-S4 and OPA1-S5).
The method of claim 2, wherein said large isoforms comprise an isoform having an apparent molecular weight of 97 kD (OPA1-L1) or an isoform having an apparent molecular weight of 92 kD (OPA1-L2), said molecular weights being determined by SDS-PAGE analysis.
The method of claim 2, wherein said small isoforms comprise an isoform having an apparent molecular weight of 88 kD (OPA1-S3), an isoform having an apparent molecular weight of 84 kD (OPA1-S4) or an isoform having an apparent molecular weight of 81 kD (OPA1-S5), said molecular weights being determined by SDS-PAGE analysis.
The method of claim 2, wherein said large isoforms comprise an isoform having an apparent molecular weight of 104 kD (OPA1-L1) or an isoform having an apparent molecular weight of 99 kD (OPA1-L2), said molecular weights being determined by mass spectrometry.
The method of claim 2, wherein said small isoforms comprise an isoform having an apparent molecular weight of 92 kD (OPA1-S3), an isoform having an apparent molecular weight of 89 kD (OPA1-S4) or an isoform having an apparent molecular weight of 87 kD (OPA1-S5), said molecular weights being determined by mass spectrometry.
The method of anyone of claims 2 to 6, wherein
said OPA1-L1 has an apparent molecular weight of 97 kD,
said OPA1-L2 has an apparent molecular weight of 92 kD,
said OPA1-S3 has an apparent molecular weight of 88 kD,
said OPA1-S4 has an apparent molecular weight of 84 kD, and/or
said OPA1-S5 has an apparent molecular weight of 81 kD,
said molecular weights being determined by SDS-PAGE analysis; or
wherein
said OPA1-L1 has an apparent molecular weight of 104 kD,
said OPA1-L2 has an apparent molecular weight of 99 kD,
said OPA1-S3 has an apparent molecular weight of 92 kD,
said OPA1-S4 has an apparent molecular weight of 89 kD, and/or
said OPA1-S5 has an apparent molecular weight of 87 kD,
said molecular weights being determined by mass spectrometry.
The method of any one of claims 1 to 7, wherein said large isoform(s) is (are) OPA1-L1 and/or OPA1-L2.
The method of any one of claims 1 to 7, wherein said small isoform(s) is (are) OPA1-S3, OPA1-S4 and/or OPA1-S5.
The method of any one of claims 2 to 9, wherein
(a) OPA1-L1 and OPA1-L2 are characterized by comprising amino acid stretches or amino acid peptides comprising one or more of the following sequences:
YLILGSAVGGGYTAK (SEQ ID No: 17);

TFDQWK (SEQ ID No: 18);

DMIPDLSEYK (SEQ ID No: 19);

WIVPDIVWEIDEYIDFEK (SEQ ID No: 20);

LAPDFDK (SEQ ID No: 21);

IVESLSLLK (SEQ ID No: 22);

ALPNSEDLVK (SEQ ID No: 23);

DFFTSGSPEETAFR (SEQ ID No: 24);

TRLLKLRYLILGS (SEQ ID No: 25); and

FWPARLATRLLKLRYLILGS (SEQ ID NO: 111).

(b) OPA1-S3 is characterized by comprising amino acid stretches or amino acid peptides comprising one or more of the following sequences:
IVESLSLLK (SEQ ID No: 22);

DFFTSGSPEETAFR (SEQ ID No: 24);

GLLGELILLQQQIQEHEEEAR (SEQ ID No: 26);

AAGQYSTSYAQQK (SEQ ID No: 27); and

IDQLQEELLHTQLK (SEQ ID No: 28).

(c) OPA1-S4 is characterized by comprising amino acid stretches or amino acid peptides comprising one or more of the following sequences:
GLLGELILLQQQIQEHEEEAR (SEQ ID No: 26);

AAGQYSTSYAQQK (SEQ ID No: 27); and

IDQLQEELLHTQLK (SEQ ID No: 28),

and/or

(d) OPA1-S5 is characterized by comprising amino acid stretches or amino acid peptides comprising the following sequence:
IDQLQEELLHTQLK (SEQ ID No: 28).
The method of any one of claims 2 to 10, wherein
(a) OPA1-L2 is characterized by not comprising amino acid stretches or amino acid peptides comprising one or more of the following sequences:
GLLGELILLQQQIQEHEEEAR (SEQ ID No: 26); and

AAGQYSTSYAQQK (SEQ ID No: 27),

and/or

(b) OPA1-S3 is characterized by not comprising amino acid stretches or amino acid peptides comprising one or more of the following sequences:
YLILGSAVGGGYTAK (SEQ ID No: 17);

TFDQWK (SEQ ID No: 18);

DMIPDLSEYK (SEQ ID No: 19);

WIVPDIVWEIDEYIDFEK (SEQ ID No: 20);

LAPDFDK (SEQ ID No: 21);

ALPNSEDLVK (SEQ ID No: 23);

FWPARLATRLLKLRYLILGS (SEQ ID NO: 111); and

TRLLKLRYLILGS (SEQ ID No: 25),

(c) OPA1-S4 is characterized by not comprising amino acid stretches or amino acid peptides comprising one or more of the following sequences:
YLILGSAVGGGYTAK (SEQ ID No: 17);

TFDQWK (SEQ ID No: 18);

DMIPDLSEYK (SEQ ID No: 19);

WIVPDIVWEIDEYIDFEK (SEQ ID No: 20);

LAPDFDK (SEQ ID No: 21);

IVESLSLLK (SEQ ID No: 22);

ALPNSEDLVK (SEQ ID No: 23);

DFFTSGSPEETAFR (SEQ ID No: 24);

FWPARLATRLLKLRYLILGS (SEQ ID NO: 111); and

TRLLKLRYLILGS (SEQ ID No: 25),

and/or

(d) OPA1-S5 is characterized by not comprising amino acid stretches or amino acid peptides comprising one or more of the following sequences:
YLILGSAVGGGYTAK (SEQ ID No: 17);

TFDQWK (SEQ ID No: 18);

DMIPDLSEYK (SEQ ID No: 19);

WIVPDIVWEIDEYIDFEK (SEQ ID No: 20);

LAPDFDK (SEQ ID No: 21);

IVESLSLLK (SEQ ID No: 22);

ALPNSEDLVK (SEQ ID No: 23);

DFFTSGSPEETAFR (SEQ ID No: 24);

TRLLKLRYLILGS (SEQ ID No: 25);

GLLGELILLQQQIQEHEEEAR (SEQ ID No: 26); and

AAGQYSTSYAQQK (SEQ ID No: 27).
The method of claims 1 to 11, wherein said determination of the ratio of OPA1 isoforms comprises
(a) the determination of the ratio of at least one large OPA1 isoform to at least one small OPA1 isoform;

(b) the determination of the ratio of at least one small OPA1 isoform to at least one large OPA1 isoform;

(c) the determination of the ratio of one OPA1 isoform to five or more OPA1 isoforms;

(d) the determination of the ratio of one OPA1 isoform to four OPA1 isoforms;

(e) the determination of the ratio of two OPA1 isoforms to three OPA1 isoforms;

(f) the determination of the ratio of three OPA1 isoforms to two OPA1 isoforms;

(g) the determination of the ratio of four OPA1 isoforms to one OPA1 isoforms; or

(h) the determination of the ratio of five or more OPA1 isoforms to one OPA1 isoforms.
The method of claims 1 to 12, whereby the ratio between at least one large OPA1 isoform and at least one small isoform is determined.
The method of claims 1 to 13, wherein the ratio between at least one small OPA1 isoform and
(a) five or more OPA1 isoforms;

(b) at least four OPA1 isoforms;

(c) at least three OPA1 isoforms;

(d) at least two OPA1 isoforms; or

(e) at least one OPA1 isoforms is determined.
The method of claim 14, wherein the ratio between the small OPA1-S5 as defined in any one of claims 4, 6, 7, 10 or 11 and at least one other OPA1 isoform as defined in any one of claims 3 to 7 and 10 or 11 is determined.
The method of claim 14 or 15, wherein the ratio between the small OPA1-S5 as defined in any one of claims 4, 6, 7, 10 or 11 and all five OPA1 isoforms as defined in any one of claims 3 to 7 and 10 or 11 is determined.
The method of any one of claims 1 to 16, wherein said method for determining the susceptibility, predisposition or the presence of a disorder correlated with mitochondrial dysfunction or disease, comprising the in vitro determination of the ratio of large and small isoforms of OPA1 in a given sample comprises a comparison of the obtained data/values to data/values obtained from a healthy control/standard and/or a comparison of the obtained data/values to data/values obtained from a known diseased control/standard and/or a comparison of the obtained data/values to data/values obtained for an internal standard.
The method of claims 1 to 11 and 17, wherein said internal standard is β-actin.
The method of any one of claims 2 to 18, said method comprising the steps of determing in a sample the ratio of at least two OPA1 isoforms selected from the group consisting of OPA1-L1, -L2,- S3, -S4 and -S5,
wherein said ratio of said isoforms is compared to the corresponding isoforms of a healthy control or normal standard.
The method of any one of claims 1 to 19, wherein said disorder is selected from the group consisting of premature ageing, cardiomyopathy, a respiratory chain disorder, mtDNA depletion syndrome, myoclonus epilepsy, ragged-red fibers syndrome (MERRF), myopathy encephalopathy lactic acidosis, stroke-like episodes (MELAS) and optic atrophy.